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2-D Electrophoresis - GE Healthcare Life Sciences

1. Final concentration Amount Urea FW 60 06 7M 10 59 Thiourea FW 76 12 2M 3 8g CHAPS 4 w v 1 0g Pharmalytet or IPG Buffert 2 v v 500 ul DTT FW 154 2 40 mM 154 mg Double distilled water to 25 ml 13 5 ml required Other neutral or zwitterionic detergents may be used at concentrations up to 2 w v Examples include Triton X 100 NP 40 octyl glucoside and the alkylamidosulfobetaine detergents ASB 14 and ASB 16 Calbiochem t Carrier ampholytes Pharmalyte or IPG buffer should be excluded from the sample extraction solution if the samples are to be labeled using 2 D DIGE Use IPG Buffer in the pH range corresponding to the pH range of the IEF separation to be performed or Pharmalyte in a pH range approximating the pH range of the IEF separation to be performed Store in 2 5 ml aliquots at 20 C 80 6429 60 AD 129 C Urea rehydration stock solution 8 M urea 2 CHAPS 0 5 2 Pharmalyte or IPG Buffer 0 002 bromophenol blue 25 ml Final concentration Amount Urea FW 60 06 8 Mt 12g CHAPS 2 w v 0 5g Pharmalyte or IPG Buffer same range as the IPG strip 0 5 v v or 2 v v 125 ul or 500 ul 1 Bromophenol blue stock solution 0 002 50 ul Double distilled water to 25 ml 16 ml required DTT is added just prior to use 7 mg DTT per 2 5 ml aliquot of rehydration stock solution For rehydration loading sample is also added to the aliquot of rehydration soluti
2. pH intervals Step Voltage V Time h kVh 3 10 1 500 0 01 3 11 NL 2 3500 1 30 3 0 6 11 3 3500 4 50 6 20 17 0 22 0 Total 6 20 7 50 20 0 25 0 4 7 1 500 0 01 3 10 NL 2 500 6 00 3 0 3 5 6 NL 3 3500 1 30 3 0 4 3500 25 9 25 19 0 30 0 Total 12 55 16 55 25 0 36 0 6 9 1 500 0 01 7 11 NL an 500 3 00 15 3 3500 1 30 3 0 4 3500 10 10 13 00 35 5 45 5 Total 14 40 17 30 40 0 50 0 5 3 6 5 1 500 0 01 6 2 7 5 2 3500 1 30 3 0 3 3500 19 10 23 25 67 0 82 0 Total 20 40 24 55 70 0 85 0 This step is added to give a convenient overnight run 15 h This step may be omitted Step 4 should then be extended by 2 5 kVh 98 80 6429 60 AD 24 cm strips pH intervals Step Voltage V Duration h min kVh 3 11 NL 1 500 0 01 3 10 2 500 5 00 2 5 3 3500 1 30 3 0 4 3500 8 30 11 20 29 5 39 5 otal 15 00 17 50 35 0 45 0 3 10 NL 1 500 0 01 4 7 2 3500 1 30 3 0 3 7 NL 3 3500 12 00 16 20 42 0 57 0 3 5 6 NL Total 13 30 17 50 45 0 60 0 6 9 500 0 01 7 11 NL 2 3500 1 30 3 0 3 3500 16 20 22 00 57 0 77 0 Total 17 50 23 30 60 0 80 0 3 5 4 5 500 0 01 2 3500 1 30 3 0 3 3500 22 00 27 40 77 0 97 0 Total 23 30 29 10 80 0 100 0 5 3 6 5 500 0 01 6 2 7 5 2 3500 1 30 3 0 3 3500 30 35 36 20 107 0 127 0 Total 32 06 37 50 110 0 130 0 This step is added to give a convenient overnight run 15 h This step can be omitted Step 4 should then be extended by 2 5 kVh 4 1 8 Preservation of focused Immobiline DryStrip gels After IEF is co
3. Proteins may be difficult to resolubilize and may not resolubilize completely Extended exposure to this low pH solution may cause some protein degradation or modification continues on following page 80 6429 60 AD 25 Table 8 Precipitation procedures continued Precipitation method General procedure Limitations Precipitation with ammonium acetate in methanol following phenol extraction This technique has proven useful Proteins in the sample are extracted into The method is complicated and with plant samples containing high water or buffer saturated phenol Proteins time consuming levels of interfering substances are precipitated from the phenol phase with 0 1 M ammonium acetate in methanol The pellet is washed several times with ammonium acetate in methanol and then with acetone Residual acetone is evaporated 42 43 47 53 For an overview of precipitation techniques see references 17 18 and 44 1 4 1 Cleaning up samples using 2 D Clean Up Kit 2 D Clean Up Kit is designed to prepare samples that would otherwise produce poor 2 D results due to high conductivity high levels of interfering substances or low concentration of protein Current methods of protein precipitation suffer from several significant disadvantages e Precipitation can be incomplete resulting in the loss of proteins from the sample and introduction of bias into the 2 D result e The precipitated protein can be difficult
4. For free floating gels remove the gel from the plates by floating the gel off with gentle agitation in the fix solution For backed gels place the gel and plate directly into the fix solution Note Place only one gel in each container The stacking gel can be left attached to help with gel orientation 3 Incubate in the fixation solution A for a minimum of 1 hour at room temperature with gentle agitation Note Overnight fixation should be used for backed gels large format gels and thick gels gt 1 5 mm and it is also recommended for applications where maximum sensitivity is required Staining 1 Take the stain out of the freezer 15 to 30 C and allow to stand at room temperature for 15 30 min Prepare working stain solution B 2 Pour off the fixation solution and replace with working stain solution B in 20 fold excess Try to minimize carry over of the acidic fixation solution Stain for 1 hour 1 0 mm thick gels For gels 1 5 mm thick or backed gels increase the staining time to 1 5 hours Extending the staining time up to 2 hours will not adversely affect results There will be some loss in fluorescence intensity if the staining time is greater than 3 hours Note The staining solution degrades over time and should not be stored If the total staining procedure is extended to more than eight hours containers can be wrapped in foil or covered with black plastic Alternatively containers and lids of a solid colored plastic
5. Perdew G H et al The use of a zwitterionic detergent in two dimensional gel electrophoresis of trout liver microsomes Anal Biochem 135 453 455 1983 Rabilloud T et al Analysis of membrane proteins by two dimensional electrophoresis comparison of the proteins extracted from normal or Plasmodium falciparum infected erythrocyte ghosts Electrophoresis 20 3603 3610 1999 146 80 6429 60 AD 60 6 6 63 64 65 66 6 68 69 70 7 7 13 74 7 76 T 78 79 80 8 8 83 84 8 nn 86 8 i fu p Ps o 3 Ee m Santoni V et al Membrane proteins and proteomics un amour impossible Electrophoresis 21 1054 1070 2000 Wilson D L et al Some improvements in two dimensional gel electrophoresis of proteins Anal Biochem 83 33 44 1977 Olsson I et al Organic disulfides as a means to generate streak free two dimensional maps with narrow range basic immobilized pH gradient strips as first dimension Proteomics 2 1630 1632 2002 Herbert B R et al Improved protein solubility in two dimensional electrophoresis using tributyl phosphine as reducing agent Electrophoresis 19 845 851 1998 Wildgruber W et al Web based two dimensional database of Saccharomyces cerevisiae proteins using immobilized pH gradients rom pH 6 to pH 12 and matrix assisted laser desorption ionization time of flight mass spectrometry Pr
6. Strip length IPG Strips 24cm 7cm Narrow Fig 18 A comprehensive range of overlapping IPG strips covering narrow medium and wide pH ranges are available in several different strip lengths DryStrip gels are rehydrated in a solution containing the necessary additives and optionally the sample proteins rehydration solution is described in detail in section 2 6 IEF is performed at high voltage After IEF the Immobiline DryStrip gels are equilibrated in equilibration solution and applied onto vertical or flatbed SDS polyacrylamide gels for the second dimension separation see chapter 3 w After IEF proceed to the second dimension separation immediately or store the Immobiline DryStrip gels at 60 C or below as described in section 2 8 3 2 2 1 Choosing strip length Immobiline DryStrip gels are available with strip lengths of 7 11 13 18 and 24 cm with a precise gel length tolerance of 2 mm Choose shorter strips i e up to 13 cm for fast cost effective screening or when the most abundant proteins are of highest interest as in prefractionated protein complexes The shortest IPG strips give the fastest results but their sample load is limited Use longer strips i e 18 and 24 cm strips for maximal resolution and loading capacity Longer strips allow detection of more spots and make it easier to select and identify the proteins in the map but require longer times in both the first and the second dimension separ
7. Use at concentrations up to 1 mM PMSF is an irreversible inhibitor that inactivates e serine proteases e some cysteine proteases PMSF rapidly becomes inactive in aqueous solutions Prepare just prior to use PMSF may be less effective in the presence of thiol reagents such as DTT or 2 mercaptoethanol This limitation can be overcome by disrupting the sample into PMSF containing solution lacking thiol reagents Thiol reagents can be added at a later stage PMSF is very toxic AEBSF Aminoethyl benzylsulfony fluoride or Pefabloc SC Serine Protease Inhibitor Use at concentrations up to 4 mM AEBSF is similar to PMSF in its inhibitory activity but is more soluble and less toxic AEBSF induced modifications can potentially alter the pl of a protein EDTA or EGTA Use at 1 mM These compounds inhibit metalloproteases by chelating free metal ions required for activity Peptide protease inhibitors e g leupeptin pepstatin aprotinin bestatin e reversible inhibitors active in the presence of DTT active at low concentrations under a variety of conditions Use at 2 20 ug ml Leupeptin inhibits many serine and cysteine proteases Pepstatin inhibits aspartyl proteases e g acidic proteases such as pepsin Aprotinin inhibits many serine proteases Bestatin inhibits aminopeptidases Peptide protease inhibitors are e expensive e small peptides and thus may appear on the 2 D map depen
8. 5 5 3 3 5 v S p g S 2 200 350 400 450 500 550 650 700 Wavelength am r232nM dem280 BP30 Filters 550 4 5 S 3 3 w fad w c a fa Q 2 2 350 400 450 S00 550 600 650 700 750 Wavelength nm 1 633nm 1 670 BP30 Filters Cy5 64 p c C fsd g Black curve excitation blue curve emission for each of the dyes T 8 The numbers at the top of each curve indicate the maxima ig S Cy2 excitation wavelength Cy3 excitation wavelength 2 4 Cy5 excitation excitation wavelength X nm 0 1 nm E Cy2 emission filter Ill Cy3 emission filter E Cy5 emission filter Filters the values for each of the excitation and emission filters 400 450 500 550 600 650 700 750 800 for each dye are chosen to minimize cross talk BP40 bandpass Wavelength nm of 40 Am 20 nm BP30 bandpass of 30 A 15 nm Fig 60 Excitation and emission spectra of CyDye DIGE Fluor minimal dyes Cy2 Cy3 and Cy5 showing the excitation and emission filters 80 6429 60 AD 113 6 1 2 Minimal labeling of protein with CyDye DIGE Fluor minimal dyes CyDye DIGE Fluor minimal dyes contain an N hydroxysuccinimidyl ester reactive group This enables labeling of lysine residues within proteins forming a covalent bond with the epsilon amino group of lysine residues to yield an amide linkage Fig 61 The recommended concentration of fluor present in a protein labeling reaction ensures that the fluor is limiting This leads to the labeling of ap
9. C Apply sample after gel rehydration If the sample was not applied as a part of the rehydration solution it can be applied immediately prior to IEF 1 Prepare sample Prepare the sample in a solution similar in composition to the rehydration solution used 2 Apply sample Pipette the sample into either or both of the lateral wells at either end of the Strip Holder Fig 27 Introduce the sample below the Immobiline DryStrip Cover Fluid Up to 7 5 ul of sample solution can be added to each side i e 15 ul per well or 30 ul total if both sides of both wells are used ere The Immobiline DryStrip gel backing is impermeable do not apply the sample to the back of the strip Replace cover on Strip Holder Refer to Table 18 section 2 6 2 for rehydration solution volume per Immobiline DryStrip Fig 27 Applying sample after gel rehydration Protocol Using IPGbox for Rehydration If the Manifold is used Immobiline DryStrip gels must be rehydrated prior to IEF in the Immobiline DryStrip IPGbox Rehydration can take place with or without the sample included Wi Do not use the Manifold for rehydration Each tray has 12 independent reservoir channels that can each hold a single Immobiline DryStrip gel up to 24 cm long Separate channels allow the rehydration of individual gel strips with no danger of spillover into adjacent lanes 1 Select and prepare a rehydration solution see Section 2 6 1 2 Place the IPGbox on a levelled tab
10. v v H O hydrogen peroxide followed by a rinse with double distilled water see the user manual for full details 2 Set up the scanner as recommended in the relevant system operational manual For example the following settings are recommended for use with a Typhoon scanner Excitation Green laser 532 nm Emission 560LP or 610BP filter Pre scan using 1000 micron resolution and then scan using a 100 micron resolution Note If the pre scan shows saturated bands spots reduce the PMT voltage rating and pre scan again If the pre scan shows too low signal increase the PMT voltage rating and pre scan again Deep Purple Total Protein Stain can also be imaged on a Typhoon scanner using the blue laser 457 nm or 488 nm If using an alternate fluorescent scanner for the best optimal images scan using as similar settings as possible to those recommended 80 6429 60 AD 139 3 Process the image according to experimental requirements and the instructions for the relevant software program B Imaging with UV light sources 1 Place the gel onto the UV transilluminator 302 or 365 nm wavelength emission required and follow the operating and safety instructions as relevant for the excitation instrument and imaging system Images can be captured using appropriate camera systems and filters film video CCD Note For long periods of illumination it is advisable to place the gel on a glass plate raised on spacers above the transilluminator
11. Appendix VI Using Ready Sol PlusOne ReadySol IEF 40 T and 3 C see ordering information is a premade stock solution of acrylamide and bisacrylamide Note that the C will not change on dilution as it is a ratio The table below provides the recipe for making 100 ml of each percentage gel 5 7 5 10 12 5 15 Monomer stock solution ReadySol 40 T 3 C ml 12 5 18 75 25 0 31 25 375 4x Resolving gel buffer ml 25 0 25 0 25 0 25 0 25 0 10 SDS ml 1 0 1 0 1 0 1 0 1 0 Double distilled water ml 61 0 54 75 48 5 42 25 36 0 10 Ammonium persulfate ml 0 5 0 5 0 5 0 5 0 5 TEMED 33 ul 33 ul 33 ul 33 ul 33 ul 4x Resolving gel buffer is 1 5 M Tris base ph 8 8 To make mix 181 7 g Tris base with 750 ml double distilled water adjust the pH to 8 8 with HCI and make up to final volume of 1 with double distilled water See also solution H in appendix t Add ammonium persulfate and TEMED just before casting the gel 80 6429 60 AD 143 144 80 6429 60 AD References 1 2 10 2 jak 2 23 24 2 wn 26 27 28 29 30 13 14 15 Bollag D M et al Protein extraction in Protein Methods Wiley Liss New York 1991 fo O Farrell P H High resolution two dimensional electrophoresis of proteins J Biol Chem 250 4007 4021 1975 Bjellqvist B et al Isoelectric focusing in immobilized pH gradients principle methodology and some applicat
12. COOMASSIE Blue G 250 StOCK reseceseseessessssssssssssessecececeeceeeeesesestssstmmtutsssnesensessssssseeeeeeceeeeeeeeeeseeteesttetteet 135 Colloidal Coomassie Blue G 250 dye stock SOLUtION esssseseseesssssssssssssscccceccecessssnssssnunnunesssseseseeeee 135 Colloidal Coomassie Blue G 250 working SOLUTION cesssssssssssssssssssssesseseceeceeecesssssssssssnsutnnssecseseee 135 PUDONG I IV AEEA OE E ceeds se Giscastcasccedovcvah cosets ase sen cove ead E E A oetescasosteuse seaceteleneatvistis 137 PRD eiae IE C i ARANA OEE E E T ETEA E AAT 141 Treating glass plates With Bind Sila Es 141 AppendiX Vaa a E a e Ea EE EEEa e Ea aeS ES E E EREE Nae 143 Using Reddy SOli E ER E E EGN a 143 REPRE COS easecesscsscscstossesssxesscaseden dapecustecaacestesuesbuces sussesssdesabasavensessudessassseasasensasasensdosncisbesbscpanssansnsaanasaiatenace 145 Additional reading and reference Mate idl ccessssssscssececesssescsececececesesesececesseseseseeesteneaeseseeeeees 149 Ordering information ccsecesecteteeeeeeee Recommended additional consumables Introduction Introduction to this handbook This handbook is intended to provide guidelines for performing high resolution two dimensional 2 D electrophoresis It is divided into seven chapters Chapter 1 provides guidelines for sample preparation and protein quantitation Chapter 2 details procedures for performing the first dimension of 2 D electrophoresis hig
13. Sample cups 60 pk 18 1004 35 IEF electrode strips 100 pk 18 1004 40 See also under Second dimension products and accessories Strip Holders for use with Immobiline DryStrip and Ettan IPGphor 3 Isoelectric Focusing System 7cm 1 pk 80 6416 87 6 pk 80 6416 11 11 cm 1 pk 80 6417 06 6 pk 80 6416 30 13 cm 1 pk 80 6417 25 6 pk 80 6416 49 18 cm 1 pk 80 6417 44 6 pk 80 6416 68 24 cm 1 pk 80 6470 07 6 pk 80 6469 88 Immobiline DryStrip Cover Fluid Il 17 1335 01 Cleaning Solution Strip Holder 950 ml 80 6452 78 80 6429 60 AD 151 Product Quantity Code No Immobiline DryStrip gels all 12 pk 7cm pH 3 5 6 NL 17 6003 53 pH 3 10 17 6001 11 pH 3 10 NL 17 6001 12 pH 3 11 NL 17 6003 73 pH 5 3 6 5 17 6003 58 pH 6 2 7 5 17 6003 63 pH 4 7 17 6001 10 pH 6 11 17 6001 94 pH 7 11 NL 17 6003 68 11 cm pH 3 5 6 NL 17 6003 54 pH 3 10 18 1016 61 pH 3 11 NL 17 6003 74 pH 4 7 18 1016 60 pH 5 3 6 5 17 6003 59 pH 6 11 17 6001 95 pH 6 2 7 5 17 6003 64 pH 7 11 NL 17 6003 69 13 cm PH 3 5 6 NL 7 6003 55 pH 3 10 17 6001 14 pH 3 10 NL 17 6001 15 pH 3 11 NL 7 6003 75 pH 4 7 7 6001 13 PH 5 3 6 5 17 6003 60 pH 6 11 17 6001 96 PH 6 2 7 5 7 6003 65 pH 7 11 NL 7 6003 70 18cm pH 3 5 6 NL 17 6003 56 pH 3 10 17 1234 01 pH 3 10 NL 17 1235 01 pH 3 11 NL 17 6003 76 pH 4 7 17 1233 01 pH 6 11 17 6001 97 pH 6 9 17 6001 88 pH 6 2 7 5 17 6003 66 pH 7 11 NL 17 6003 71 24 cm pH 3 5 6 NL 17 6003 57 pH 3 10 17 6002 44 pH 3 10 NL 17 6002 45 pH 3 1
14. The anode assembly is molded so that it can only be inserted in one orientation The side edge of the assembly should fit into the slot in the side of the tank 3 Fill with anode buffer Pour the diluted anode buffer into the tank of the Ettan DALTsix Electrophoresis System Fig 34 Switch on the pump 4 Switch on the temperature controller Switch on the MultiTemp III temperature controller and adjust the temperature to the desired setting A temperature of 25 C is recommended for electrophoresis 5 Set aside upper chamber The upper chamber is prepared once the gel has been inserted into Ettan Daltsix See section 3 3 5 74 80 6429 60 AD Fig 34 Filling the Ettan DALTsix electrophoresis unit with anode buffer Protocol Preparing Ettan DALTtwelve For detailed instructions for using Ettan DALTtwelve system please refer to the Ettan DALTtwelve user manual 1 Prepare cathode buffer Dilute the cathode buffer included in the DALT Buffer Kit to working strength by adding both bottles of 10x cathode buffer total volume 250 ml to 2 25 distilled or deionized water 2 Prepare anode buffer Ensuring that the valve on the separation unit is set to circulate fill the tank to the 7 5 fill line with distilled or deionized water Add the entire contents 75 ml of the 100x anode buffer included in the DALT Buffer Kit into the tank Avoid pouring the 100x anode buffer on the tubing by spreading the tubing slightly with one
15. and the file outputs are separated automatically in a format that is compatible with DeCyder 2 D Differential Analysis Software The gels can be easily scanned between low fluorescence glass plates which prevents drying and shrinkage and also allows for further running and scanning For additional information relating to the use of the Typhoon Variable Mode Imager for the Ettan DIGE system refer to the Ettan DIGE user manual 122 80 6429 60 AD 6 3 7 Image analysis with DeCyder 2 D Differential Analysis Software DeCyder 2 D Differential Analysis Software developed to exploit the advantages of CyDye DIGE Fluor dyes consists of a fully automated image analysis software suite that enables the detection quantitation matching and analysis of gels used with Ettan DIGE system The co detection algorithm in DeCyder 2 D software co detects overlaid image pairs and produces identical spot boundaries for each pair This enables direct spot volume ratio measurements and therefore produces an accurate comparison of every protein with its representative in gel internal standard The software automatically performs detection background subtraction quantitation and normalization which takes into account any differences in the dyes i e molar extinction co efficients quantum yields etc These steps can be broken into the following processes which form part of the built in functionality of DeCyder 2 D software and are performed automatically
16. preparative gel for spot picking The gel must be stained using a fluorescent post stain to allow matching to analytical gels for picking 6 3 6 Imaging Typhoon is a highly sensitive variable mode imager that has been adapted to meet the specific needs of 2 D DIGE Typhoon Variable Mode Imager optimally detects Cy2 Cy3 and Cy5 signals with exceptional signal to noise ratio due to consistent point light illumination that eliminates the need for image stitching confocal optics that exclude artifacts and narrow band pass filters that maximize signal to noise ratio Fig 60 A linear protein concentration response over five orders of magnitude is possible with Ettan DIGE system 96 compared with silver which has a dynamic range of less than two orders of magnitude 97 In addition silver stained proteins saturate more readily which produces data that cannot be accurately quantitated The wide dynamic range provided by CyDye 2 D DIGE Fluor minimal dyes in combination with the Typhoon Variable Mode Imager enables production of data that is quantitative and reproducible and that offers a sensitivity down to 125 pg protein compared with approximately 5 ng for silver staining 98 Specific gel alignment guides enable the correct positioning of both DALT and SE 600 Ruby gels on the scanner to simplify gel handling and to reduce hands on time Two large format DALT gels or four SE 600 Ruby gels can be imaged simultaneously
17. 0 40 1 05 2 2 3 7 Total 2 10 2 35 5 0 6 5 3 10 NL 200 0 01 4 7 2 3500 1 30 2 8 3 5 6 NL 3 3500 0 55 1 30 3 2 5 2 Total 2 25 3 00 6 0 8 0 7 11 NL 300 0 01 2 3500 1 30 2 9 3 3500 1 10 2 02 4 1 7 1 Total 2 40 3 30 7 0 10 0 5 3 6 5 300 0 01 6 2 7 5 2 3500 1 30 2 9 3 3500 2 36 3 45 9 1 13 1 Total 4 06 5 15 12 0 16 0 11 cm strips pH intervals Step Voltage V Time h kVh 3 11 NL 1 300 0 01 3 10 2 3500 1 30 2 9 6 11 3 3500 1 45 2 35 6 1 9 1 Total 3 15 4 05 9 0 12 0 4 7 300 0 01 3 5 6 NL 2 3500 1 30 2 9 3 3500 2 20 3 30 8 1 12 1 Total 3 50 5 00 11 0 15 0 7 11 NL 300 0 01 2 3500 1 30 2 9 3 3500 3 30 4 55 12 1 17 1 Total 5 00 6 25 15 0 20 0 5 3 6 5 1 500 0 01 6 2 7 5 2 3500 1 30 3 0 3 3500 7 10 9 10 25 0 32 0 Total 8 40 10 40 28 0 35 0 To adjust this protocol for an overnight run extend step 1 by 5 h 2 5 kVh and reduce step 3 by 2 5 kVh 80 6429 60 AD 97 13 cm strips pH intervals Step Voltage V Time h kVh 3 10 A 300 0 01 3 11 NL 2 3500 1 30 2 9 6 11 3 3500 3 10 4 00 11 1 14 1 Total 4 40 5 30 14 0 17 0 3 10 NL 1 300 0 01 4 7 2 3500 1 30 2 9 3 5 6 NL 3 3500 3 45 5 10 13 1 18 1 Total 5 15 6 40 16 0 21 0 7 11 NL 1 500 0 01 2 3500 1 30 3 0 3 3500 5 10 6 20 18 1 22 0 Total 6 40 7 50 21 0 25 0 5 3 6 5 1 500 0 01 6 2 7 5 2 3500 1 30 3 0 3 3500 10 00 12 50 35 0 45 0 Total 11 30 14 20 38 0 48 0 To adjust this protocol for an overnight run extend the time of step 1 to 2h 18 cm strips
18. 0 5 3 5 6 NL 2 Gradient 1000 1 00 0 8 3 Gradient 6000 2 30 8 8 4 Step and Hold 6000 0 10 0 50 0 9 4 9 Total 4 40 5 20 11 0 15 0 7 11 NL 1 Step and Hold 500 1 00 0 5 2 Gradient 1000 1 00 0 8 3 Gradient 6000 2 30 8 8 4 Step and Hold 6000 0 50 1 40 4 9 9 9 Total 5 20 6 10 15 0 20 0 53 65 1 Step and Hold 500 1 00 0 5 6 2 7 5 2 Gradient 1000 1 00 0 8 3 Gradient 6000 3 00 10 5 4 Step and Hold 6000 2 40 3 50 16 2 23 2 Total 7 40 8 50 28 0 35 0 To convert this to a convenient overnight run extend Step 1 to 6 h 3 kVh and reduce step 4 by 3 kVh 13 cm strips pH Step Voltage Voltage Time kVh intervals mode Vv h min 3 10 1 Step and Hold 500 1 00 0 5 3 11 NL 2 Gradient 1000 1 00 0 8 6 11 3 Gradient 8000 2 30 11 3 4 Step and Hold 8000 0 10 0 30 1 4 4 4 Total 4 40 5 00 14 0 17 0 3 10 NL 1 Step and Hold 500 1 00 0 5 4 7 2 Gradient 1000 1 00 0 8 3 5 6 NL 3 Gradient 8000 2 30 113 4 Step and Hold 8000 0 25 0 55 3 4 7 4 Total 4 55 5 25 16 0 20 0 7 11 NL 1 Step and Hold 500 1 00 0 5 2 Gradient 1000 1 00 0 8 3 Gradient 8000 3 00 13 5 4 Step and Hold 8000 0 45 1 15 6 2 10 2 Total 5 45 6 15 21 0 25 0 53 65 1 Step and Hold 500 1 00 0 5 6 2 7 5 2 Gradient 1000 1 00 0 8 3 Gradient 8000 3 00 13 5 4 Step and Hold 8000 2 55 4 10 23 2 33 2 Total 7 55 9 10 38 0 48 0 To convert this to a convenient overnight run extend Step 1 to 6 h 3 kVh and reduce step 4 by 3 kVh 80 6429 60 AD 65 Table 19 continued 18 cm strips Note When using
19. 0 mm gels 1x SDS electrophoresis buffer 43 2x SDS electrophoresis buffer 800 1 5 mm gels 1x SDS electrophoresis buffer 43 3x SDS electrophoresis buffer 800 Ettan DALTtwelve 1 0 or 1 5 mm gels 1x SDS electrophoresis buffer 75 2x SDS electrophoresis buffer 2500 Protocol Preparing Ettan DALTsix for use Fill the electrophoresis tank with 4 3 of 1x SDS electrophoresis buffer 2 Turn onthe pump 3 Switch on the MultiTemp IIl temperature controller and set the desired temperature ET A temperature of 10 C is recommended for rapid electrophoresis Equilibrate the buffer temperature to at least 15 C before starting the run Protocol Preparing Ettan DALTtwelve for use Fill the anodic chambers of the tank with 1x SDS electrophoresis buffer 1 Set the valve on the separation unit to circulate Fill the tank to the 7 5 I fill line with 1x SDS electrophoresis buffer 2 Switch on the separation unit 3 Turn on the pump to mix the buffers and set the separation unit to desired temperature Er A temperature of 25 C is recommended for rapid electrophoresis 3 3 9 Equilibrating Immobiline DryStrip gels with lab cast gels When the buffer tank has reached the desired temperature start equilibrating the Immobiline DryStrip gel as described in section 3 1 2 The equilibration procedure is the same whether applying the strip to precast or lab cast gels 3 3 10 Applying Immobiline DryStrip gels to lab cast gels To apply the
20. 1 Sample preparation 2 Sample labeling with CyDye DIGE fluors 3 Addition of DTT and ampholytes Proteins are extracted from cells or tis Size and charge matched spectrally resolvable Following sample labeling DTT and am sues of interest dyes enable simultaneous co separation and pholytes are added to each sample analysis of samples on a single gel multiplexing J a CHAPS gem A qm DTT i gt A A Teen ii Thiourea Urea CHAPS Tris butfer pH 8 5 4 2 D electrophoresis 5 Image acquisition 6 Image analysis Up to three samples one of which is The gel is scanned using the highly sensitive DeCyder software automatically locates and analyzes the internal pooled standard can be simul Typhoon FLA 9000 optimized for Ettan DIGE multiplexed samples in a gel within minutes It allows taneously separated on a single Perpendicular scanning assures quantitation complex analysis of multiple gels to provide comparative 2 D gel using IPGphor or Multiphor II of protein expression levels and identification and accurate measurement of differential protein in the first dimension and Ettan DALT of spots with high precision expression in the second dimension Fig 59 Ettan DIGE system for protein analysis Protein samples are extracted 1 and labeled with CyDye DIGE Fluor minimal dyes in the absence of DTT and ampholytes 2 These are added following the labeling reaction 3 The fluors enable up to two samples plus an
21. 10 h 92 80 6429 60 AD 4 1 2 Preparing for IEF The components of the Immobiline DryStrip Kit include a tray and electrode holder anode and cathode electrodes an Immobiline DryStrip aligner a sample cup bar and sample cups EF Procedures A and B below should be completed before the Immobiline DryStrip gels are removed from the Immobiline DryStrip IPGbox A Prepare the Immobiline DryStrip Kit la Clean all components of the Immobiline DryStrip Kit The Immobiline DryStrip tray Immobiline DryStrip aligner electrodes sample cup bar and sample cups must be clean and ready for use Clean with detergent rinse thoroughly with distilled water and allow to dry Confirm electrical connections on Multiphor II Electrophoresis System Check that the red bridging cable in the Multiphor II unit is connected seated under the cooling plate Establish cooling Set the temperature on MultiTemp IIl Thermostatic Circulator to 20 C Position the cooling plate on the Multiphor II unit and ensure that the surface is level Position the Immobiline DryStrip tray Pipette approximately 3 4 ml of Immobiline DryStrip Cover Fluid onto the cooling plate Position the Immobiline DryStrip tray on the cooling plate so the red anodic electrode connection of the tray is positioned at the top of the plate near the cooling tubes Remove any large bubbles between the tray and the cooling plate small bubbles can be ignored Immobiline DryStrip Cover Flui
22. 1996 Cull M and McHenry C S Preparation of extracts from prokaryotes Methods Enzymol 182 147 153 1990 Jazwinski S M Preparation of extracts from yeast Methods Enzymol 182 154 174 1990 Kawaguchi S I and Kuramitsu S Separation of heat stable proteins from Thermus thermophilus HB8 by two dimensional electrophoresis Electrophoresis 16 1060 1066 1995 Teixeira Gomes A P et al Mapping and identification of Brucella melitensis proteins by two dimensional electrophoresis and microsequencing Electrophoresis 18 156 162 1997 Ames G F L and Nikaido K Two dimensional gel electrophoresis of membrane proteins Biochemistry 15 616 623 1976 Gorg A et al Two dimensional electrophoresis with immobilized pH gradients of leaf proteins from barley Hordeum vulgare method reproducibility and genetic aspects Electrophoresis 9 681 692 1988 Posch A et al Genetic variability of carrot seed proteins analyzed by one and two dimensional electrophoresis with immobilized pH gradients Electrophoresis 16 1312 1316 1995 Geigenheimer P Preparation of extracts from plants Methods Enzymol 182 174 193 1990 80 6429 60 AD 145 31 3 33 34 35 36 3 N 38 59 40 4 4 43 44 4 46 4 48 49 50 5 5 53 54 55 56 57 58 59 i ya fu pn ps Py Theillet C et al Influence of the ex
23. 9 add 150 ul DeStreak rehydration solution to the cathodic wick The electrode must contact the wick With the electrode cams in the open position place the electrode assembly on top of all the wicks Swivel the cams into the closed position under the external lip of the tray The electrodes should not be moved while the cams are in the closed position Fig 33 Briefly centrifuge the protein sample e g at top speed in a microcentrifuge prior to loading to remove insoluble material and particulate matter These materials could impede sample entry and result in vertical streaks in the second dimension gel Load samples into the sample cups A maximum of 150 ul of sample may be placed in these cups Check to make sure that there is cover fluid over the samples When the cups are initially placed on the Manifold cover fluid will flow into the cups as they are seated When sample is introduced into the cups the sample will sink to the bottom of the cup and contact the IPG strip 80 6429 60AD 61 Note For basic IPG strips superior focusing patterns are generally obtained when the sample cup is placed as close to the anodic electrode as possible 6 Close the Ettan IPGphor 3 lid Program the Ettan IPGphor 3 with the desired run parameters Ramping the voltage slowly while the sample is entering the IPG strip will improve results See section 2 8 for further discussion on this topic Optimal ramp voltages and times or Vhr volt hours totals mus
24. ASB 14 and ASB 16 Calbiochem A Pharmalyte IPG Buffer concentration of 0 5 125 ul is recommended with Ettan IPGphor 3 Isoelectric Focusing System and an IPG Buffer Pharmalyte concentration of 2 500 ul is recommended with the Multiphor II and Immobiline DryStrip Kit system Store in 2 5 ml aliquots at 20 C E SDS equilibration buffer solution 6 M urea 75 mM Tris HCl pH 8 8 29 3 glycerol 2 SDS 0 002 bromophenol blue 200 ml Final concentration Amount Urea FW 60 06 6M 721g Tris HCI pH 8 8 see solution H 75 mM 10 0 ml Glycerol 87 w w 29 3 v v 69 ml 84 2 g SDS FW 288 38 2 w v 409g 1 Bromophenol blue stock solution 0 002 w v 400 pl Double distilled water to 200 ml This is a stock solution Just prior to use add DTT or iodoacetamide for first or second equilibration respectively as described in the protocol in section 3 1 2 Store in 20 or 50 ml aliquots at 20 C 130 80 6429 60 AD F 10x Laemmli SDS electrophoresis buffer 250 mM Tris base 1 92 M glycine 1 SDS 10 Final concentration Amount Tris base FW 121 1 250 mM 303g Glycine FW 75 07 1 92 M 14419 SDS FW 288 38 1 w v 100g Double distilled water to 101 The pH of this solution should not be adjusted Store at room temperature See also Recipe M for 1x Laemmli SDS electrophoresis buffer G 30 T 2 6 C monomer stock
25. Coomassie Blue G 250 The dye will not dissolve completely Colloidal Coomassie Blue G 250 dye stock solution 10 ammonium sulfate 1 w w phosphoric acid 0 1 Coomassie Blue G 250 500 ml Amount Ammonium sulfate FW 132 1 50g Phosphoric acid 85 w w 6 ml 5 Coomassie Blue G 250 stock 10 ml Double distilled water to 500 ml Colloidal Coomassie Blue G 250 working solution 8 ammonium sulfate 0 8 phosphoric acid 0 08 Coomassie Blue G 250 20 methanol 500 ml Amount Colloidal Coomassie Blue G 250 400 ml dye stock solution Methanol 100 ml Prepare colloidal stain immediately before staining the gel 1 Fix gel for at least 30 min in 10 acetic acid 40 ethanol 2 Decant the fixer and place the gel in colloidal stain 100 300 ml per gel depending on size 3 Leave overnight or longer The staining gets more and more intense for up to 7 days 3 Rinse gel repeatedly with water to remove residual stain 4 Soak in 5 glycerol 20 ethanol for no more than 30 min prior to drying coe The above step is necessary only if drying down the gel Ethanol will tend to shrink the gel and make it easier to handle but it will also destain the gel 80 6429 60 AD 135 136 80 6429 60 AD Appendix IV Protocol for use of Deep Purple Total Protein Stain Reagents supplied in the kit Deep Purple Total Protein Stain in 50 v v DMSO and 50 v v acetonitrile Required but not provided SDS e g PlusOne code
26. DALT Buffer Kit If using the Laemmli buffer system see appendix I solution N Melt each aliquot as needed in a 100 C heat block each gel will require 1 1 5 ml It takes approximately 10 min to fully melt the agarose a An ideal time to carry out this step is during Immobiline DryStrip gel equilibration 80 6429 60 AD 77 Allow the agarose to cool until the tube can be held with your fingers 60 C and then slowly pipette the amount required to seal the Immobiline DryStrip gel in place Fig 41 Pipette slowly to avoid introducing bubbles Apply only the minimum quantity of agarose sealing solution required to cover the Immobiline DryStrip gel Allow a minimum of 1 min for the agarose to cool and solidify Fig 39 Positioning an equilibrated Immobiline DryStrip Fig 40 Pushing the Immobiline Fig 41 Sealing the Immobiline gel on the DALT Precast Gel Cassette DryStrip gel down to contact the DryStrip gel in place on a DALT precast gel slab gel using agarose sealing solution 3 3 5 Inserting gels into Ettan DALT electrophoresis units Two protocols follow the first for inserting gels into Ettan DALTsix and the second for inserting gels into Ettan DALTtwelve Protocol Inserting gels into Ettan DALTsix When the electrophoresis buffer has reached the desired temperature insert the loaded gel cassettes with the Immobiline DryStrip gels in place 1 Insert the cassettes into the cassette carrier Fig 42 and fill any empty slots wi
27. Power delivery Current too low or zero At least two of three pressure pads on the lid adaptor of PGphor 3 under the safety lid should press gently against the strip holders to ensure electrical continuity between the strip holder electrodes and the electrode areas on the platform The gel must be evenly and completely rehydrated to conduct current Make sure the proper amount of rehydration solution is applied to the IPG strip holder and allow a minimum of 10 hours for rehydration Voltage limit not reached The ionic strength of the rehydration solution is too high reduce the IPG buffer concentration use a mixed bed ion exchange resin to remove ionic breakdown products of urea or other additives Desalt the sample or prepare the sample so that the salt concentration is less than 10 mM Sparks or burning in strips Reduce the current limit Do not exceed 50 pA per strip Prevent the IPG strip from drying out by always applying Immobiline DryStrip Cover Fluid immediately after strip placement in rehydration buffer Ensure that the IPG strip is fully rehydrated along the entire length of he strip The IPG strip should be in complete contact with the correct volume of rehydration solution Remove any air bubbles trapped under he IPG strip Desalt the sample or prepare the sample so that the salt concentration is less than 10 mM De ionize additives to the rehydration solution Excessive charged material in the sample or r
28. Silver stain Coomassie stain analytical preparative 7 3 11 NL 3 10 NL 3 10 3 6 25 60 10 4 7 4 8 25 150 13 3 5 6 NL 5 3 6 5 6 2 7 5 8 16 40 240 26 6 11 7 11 NL 11 3 11 NL 3 10 7 15 50 120 20 4 7 10 20 50 300 28 6 11 3 5 6 NL 5 3 6 5 20 40 100 600 56 6 2 7 5 7 11 NL 13 3 11 NL 3 10 NL 3 10 10 20 50 240 25 4 7 15 30 75 450 38 6 11 narrow and medium intervalst 30 60 150 900 76 18 3 11 NL 3 10 NL 3 10 20 40 100 500 50 4 7 30 60 150 900 75 6 11 6 9 narrow and medium intervals 60 120 300 1500 150 24 3 11NL 3 10 NL 3 10 30 60 200 600 100 4 7 3 7 NL 45 90 200 1300 150 6 9 narrow and medium intervals 80 200 400 2000 300 When using cup loading an increased sample concentration will lead to an increased risk of protein precipitation in the sample cup A maximum concentration of 150 ug protein 150 pl sample solution 150 pl is the volume of the cup is recommended This is a general recommendation which will function for most samples but the maximum concentration usable varies greatly between sample types For larger sample loads rehydration loading is recommended t Immobiline DryStrip gels pH intervals 3 5 6 NL 5 3 6 5 6 2 7 5 and 7 11 NL 5 Immobiline DryStrip gels pH intervals 3 5 6 NL 5 3 6 5 6 2 7 5 7 11 NL 3 5 4 5 4 0 5 0 4 5 5 5 5 0 6 0 and 5 5 6 7 2 6 Immobiline DryStrip gel rehydration solutions Immobiline DryStrip gels must be rehydrated prior to IEF They should be rehydrated in the Im
29. accurate spot statistics between gels e Increased confidence in matching between gels e Flexibility of statistical analysis depending on the relationship between samples e Separation of system variation from biological variation 118 80 6429 60 AD Table 42 shows an example of a recommended experimental setup that was designed to derive statistical data on differences between control and three treatment regimens A B and C For the control and each treatment regimen four biological replicates were included The internal standard a pool of all samples four control and 12 treated was labeled with CyDye DIGE Fluor Cy2 minimal dye and run on every gel Table 42 Setup for an Ettan DIGE system experiment Gel number cy2 Cy3 Cy5 1 Pooled standard Control 1 Sample A3 2 Pooled standard Sample A1 Sample B3 3 Pooled standard Sample B1 Sample C3 4 Pooled standard Sample C1 Control 3 5 Pooled standard Control 2 Sample B4 6 Pooled standard Sample A2 Sample C4 7 Pooled standard Sample B2 Control 4 8 Pooled standard Sample C2 Sample A4 Each gel contains CyDye DIGE Fluor Cy2 minimal dye labeled standard which is a pool of aliquots taken from each sample Four biological replicates 1 4 have been included for control and treated A B or C samples The samples have been arranged between gels to ensure an even distribution between those labeled with CyDye DIGE Fluor Cy3 minimal dye and those labeled with CyDye DIGE
30. after gel rehydration In the latter case see Note C below 1 Prepare the Strip Holder s Select the Strip Holder s corresponding to the Immobiline DryStrip gel length chosen for the experiment Wi Handle the ceramic Strip Holders with care t is essential to wash each Strip Holder with detergent to remove residual protein Use a neutral pH v detergent such as the Strip Holder Cleaning Solution to remove residual protein from the Strip Holders Strip Holder Cleaning Solution has been specifically formulated to remove protein deposits and will not damage the Strip Holder Strip Holder Cleaning Solution is available in 950 ml bottles see ordering information 1 Clean Strip Holders after each first dimension IEF run Do not let solutions dry in the Strip Holder Cleaning may be more effective if the Strip Holders are first soaked for a few hours or overnight in a solution of 2 5 Strip Holder Cleaning Solution in water First rinse off the Strip Holder to remove any residual DryStrip Cover Fluid 2 Squeeze a few drops of Strip Holder Cleaning Solution into the Strip Holder channel Use a toothbrush and vigorous agitation to clean the Strip Holder 3 Rinse well with distilled or deionized water Thoroughly air dry the Strip Holders or dry well with a lint free tissue prior to use Recalcitrant or dried on protein deposits may be removed with hot up to 95 C 1 w v SDS Add 1 w w DTT for complete removal of sticky proteins Rinse
31. all 1 45 ExcelGel Gradient KL 12 14 24 5 x 18 1 0 5 all 3 20 Multiple shorter Immobiline DryStrip gels two 11 cm strips or three 7 cm strips fit on one gel t If 1 cm wide spacers are used An accessory divider plate increases the capacity to four gels 5 Up to eight mini format separations can be simultaneously achieved using the shorter 8 cm glass plates combined with divider plates 80 6429 60 AD 13 Vertical systems Vertical systems offer relative ease of use and the possibility of performing multiple separations simultaneously Vertical 2 D gels can be either 1 or 1 5 mm thick Ettan DALTsix Fig 2 allows intermediate throughput of up to six high resolution second dimension gels The unit accommodates 18 or 24 cm Immobiline DryStrip gels that can be used with either precast or lab cast large format Ettan DALT gels A pump mounted under the lower chamber recirculates buffer around the cassettes for efficient temperature regulation in conjunction with MultiTemp III Thermostatic Circulator For maximal resolution reproducibility and capacity the large gel format of the Ettan DALTtwelve system Fig 3 is recommended Precast large format Ettan DALT gels on plastic film supports offer the convenience of ready to use gels The system can accommodate the entire length of an 18 or 24 cm Immobiline DryStrip gel plus molecular weight markers and up to 12 gels can be run simultaneously Integrated Peltier temperature contro
32. and Kobro G A method for two dimensional electrophoresis of proteins from green plant tissues Anal Biochem 177 33 36 1989 Matsui N M et al Immobilized pH gradient two dimensional gel electrophoresis and mass spectrometric identification of cytokine regulated proteins in ME 180 cervical carcinoma cells Electrophoresis 18 409 417 1997 Tsugita A et al Two dimensional electrophoresis of plant proteins and standardization of gel patterns Electrophoresis 17 855 865 1996 Gorg A et al Very alkaline immobilized pH gradients for two dimensional electrophoresis of ribosomal and nuclear proteins Electrophoresis 18 328 337 1997 Usuda H and Shimogawara K Phosphate deficiency in maize VI Changes in the two dimensional electrophoretic patterns of soluble proteins from second leaf blades associated with induced senescence Plant Cell Physiol 36 1149 1155 1995 Stasyk T et al Optimizing sample preparation for 2 D electrophoresis Life Science News 9 8 11 2001 Musante L et al Resolution of fibronectin and other uncharacterized proteins by two dimensional polyacrylamide electrophoresis with hiourea J Chromat 705 351 356 1997 Pasquali C et al Preparative two dimensional gel electrophoresis of membrane proteins Electrophoresis 18 2573 2581 1997 Rabilloud T Use of thiourea to increase the solubility of membrane proteins in two dimensional electrophoresis Electrophoresis 19 758 760 1998
33. can quantitate colorimetrically stained gels 80 6429 60 AD 107 5 3 Standardizing results 2 D electrophoresis is often used comparatively and thus requires a reproducible method for determining relative spot positions Because precast Immobiline DryStrip gels are highly reproducible the pl of a particular protein can be estimated from its focusing position along a linear pH gradient Immobiline DryStrip gel Detailed information on Immobiline DryStrip pH gradients are found in the publication Immobiline DryStrip visualization of pH gradients see additional reading and reference material The second dimension can be calibrated using molecular weight marker proteins loaded to the side of the second dimension gel Often there are abundant proteins in the sample for which the pl and molecular weight are known These proteins can serve as internal standards EF The pl of a protein is dependent on its chemical environment and can thus vary depending on the experimental conditions used The use of native pl markers is not recommended because they will run differently in a native environment compared with a denaturing environment e g urea 5 4 Further analysis of protein spots The procedure of picking and digesting spots can be performed manually or semi automatically by manual transfer of gels and microplates between the instruments as described below or fully automatically in the integrated Ettan Spot Handling Workstation Ettan Spot H
34. cm Stainless Steel Staining Tray 29 x 35 cm Blot Processing Mini Tray 3 pk Blot Processing Standard Tray 3 pk Manual gel staining Stainless Steel Staining Tray Set Staining reagents Silver Staining Kit Protein Coomassie tablets PhastGel Blue R 350 Deep Purple Total Protein Stain sufficient for two large format gels or 20 minigels Deep Purple Total Protein Stain sufficient for 10 large format gels or 100 minigels Gel dryers GD 2000 Vacuum Gel Dryer for gels up to 33 x 44 cm 115 V GD 2000 Vacuum Gel Dryer for gels up to 33 x 44 cm 230 V Cellophane Sheets Image analysis systems and software Image Scanner Ill Typhoon FLA 9000 ImageMaster 2D Platinum ImageMaster 2D Platinum site license See also under 2 D DIGE products 156 80 6429 60 AD 80 1129 83 17 0446 01 17 0615 01 RPN800 17 0582 01 80 6444 04 80 6444 80 80 6445 18 80 6444 23 80 6343 91 80 6345 24 80 6444 42 80 6444 61 80 6468 17 17 1150 01 17 0518 01 RPN6305 RPN6306 80 6428 84 80 6429 03 80 6117 81 28 9076 07 28 9558 08 18 1176 30 18 1176 31 Product Quantity Code No Spot handling Ettan Spot Picker 18 1145 28 Ettan Digester 18 1142 68 2 D DIGE products CyDye DIGE Fluor Minimal Dye Labeling Kit 25 8010 65 includes Cy2 Cy3 and Cy5 Snmol CyDye DIGE Fluor Cy2 minimal dye 5 nmol 25 8010 82 CyDye DIGE Fluor Cy2 minimal dye 10 nmol 25 8008 60 CyDye DIGE Fluor Cy2 minimal dye 2
35. dialysis e spin dialysis e gel filtration e precipitation resuspension Dialysis is a very effective method for salt removal resulting in minimal sample loss However the process is time consuming and requires large volumes of solution Spin dialysis is quicker but protein adsorption onto the dialysis membrane may be a problem Spin dialysis should be applied to samples prior to the addition of urea and detergent Gel filtration can be acceptable but often results in protein losses Precipitation resuspension is an effective means for removing salts and other contaminants but can also result in protein losses see section 1 4 Endogenous small ionic molecules nucleotides metabolites phospholipids etc Endogenous small ionic molecules are present in any cell lysate These substances are often negatively charged and can result in poor focusing toward the anode TCA acetone precipitation is particularly effective at removing this sort of contaminant Other desalting techniques may be applied see above Albumin and IgG in human serum These two major protein components of serum represent greater than 60 of the total protein in human serum content During gel analysis of serum the high concentration of albumin and IgG often masks the presence of other proteins with similar isoelectric point and or molecular weight Therefore removal of albumin and IgG from serum samples prior to electro phoresis im
36. each spot in each sample is expressed as a normalized ratio relative to spots from the in gel internal standard The same internal standard is run on all gels within the experimental series This creates an intrinsic link between internal standard and samples in each gel matching the internal standards between gels Quantitative comparisons of samples between gels are made based on the relative change of sample to its in gel internal standard This removes inter gel variation a common problem associated with traditional 2 D and separates gel to gel variation from biological variation Fig 66 enabling accurate statistical quantitation of induced biological change between samples Ettan DIGE system is currently the only protein difference analysis technique to utilize this approach 95 Gel 1 Gel 2 Sample 2 Cy5 Sample 4 Cy5 Sample 1 Cy3 Sample 3 Cy3 Matching and comparison of samples using the relative measure of sample to standard Protein N in sample 1 sample 2 sample 3 sample 4 Fig 66 Matching and comparison of samples across gels The internal standard sample present on every gel is used to aid matching of spot patterns across all gels The relative ratios of individual sample spots to their internal standards are used to accurately compare protein abundance between samples on different gels The advantages of linking every sample in gel to a common standard are e Accurate quantitation and
37. for instructions on how to program a protocol 2 8 2 Running an Ettan IPGphor 3 protocol Ensure that the Strip Holders are properly positioned on the Ettan IPGphor 3 platform Use the guide marks along the sides of the platform to position each Strip Holder and check that the pointed end of the Strip Holder is over the anode pointing to the back of the unit and the blunt end is over the cathode Please refer to the Ettan IPGphor 3 user manual for complete details Check that both external electrode contacts on the underside of each Strip Holder make metal to metal contact with the platform Before closing the safety lid insert the lid adaptor an accessory included with IPGphor 3 such that the pressure pads press gently against the cover of each Strip Holder to ensure contact between the electrodes and the electrode areas Begin IEF 80 6429 60 AD 63 EF Sa Y As isoelectric focusing proceeds the bromophenol blue tracking dye migrates toward the anode Note that he dye front leaves the Immobiline DryStrip gel well before focusing is complete so clearing of the dye is no indication that the sample is focused If the dye does not migrate no current is flowing If this occurs check the contact between the external face of the Strip Holder electrodes and the electrode areas on the instrument and between the rehydrated gel and the internal face of the electrodes Table 19 lists guidelines for running Immobiline DryStrip gels on Ettan IPGph
38. fragmentation CAF used in conjunction with MALDI MS is a method for improving fragmentation of tryptic peptides by PSD The technique introduces a negative charge at the amino terminus of the peptide Following fragmentation only y ions containing C termini are acquired in the spectra while the neutralized b ions containing N termini are not observed The spectra containing y ions are easy to interpret and amino acid sequences can be deduced by calculating the mass differences between the fragmented ions The software for automated PSD analysis has automated PSD data acquisition of selected peaks and automated PSD spectrum processing and identification This enables rapid and sensitive peptide sequencing and protein identification Chemically assisted fragmentation MALDI simplifies the amino acid sequencing of peptides and identification of phosphorylation sites 80 6429 60 AD 109 110 80 6429 60 AD 6 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE 6 0 Overview 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE is a method that labels protein samples prior to 2 D electrophoresis enabling accurate analysis of differences in protein abundance between samples 86 It is possible to separate up to three different samples within the same 2 D gel Fig 58 The technology is based on the specific properties of spectrally resolvable dyes CyDye DIGE Fluor dyes Two sets of dyes are available Cy 2 Cy3 and Cy5 mi
39. gel Refer to sections 2 4 2 7 for a discussion of rehydration and sample application methods Ettan DIGE system compatible rehydration buffer contains 7 M urea 2 M thiourea 4 CHAPS w v 1 IPG buffer or Pharmalyte v v for IEF 2 w v DTT D Separating proteins in the first dimension Ettan IPGphor 3 Isoelectric Focusing System and Multiphor II Electrophoresis System are both suitable for Ettan DIGE system applications Detailed instructions for use of the systems are given in chapters 2 and 4 respectively Ettan DIGE applications are described in detail in the Ettan DIGE user manual E Separating proteins in the second dimension Low fluorescence glass plates must be used for gels used in Ettan DIGE system Standard glass or plastic backed plates can result in the generation of a high background signal DIGE Gel is a 12 5 precast low fluorescent polyacrylamide gel cast in a low fluorescent glass cassette specially developed for 2 D DIGE analysis DIGE Gel should be used with the DIGE Buffer Kit which consists of concentrated running buffers and Sealing Solution for attaching Immobiline DryStrip Gels IPG strips to the top of the polyacrylamide gel The capacity of the buffer system is similar to the commonly used Laemmli Tris Glycine buffer system and the separation performance of DIGE Gel is comparable to 12 5 Laemmli gels DALT gels are large enough to accommodate the longest Immobiline DryStrip gels 24 cm and can be run
40. gel length e Intermediate throughput up to four gels simultaneously using divider plates e Best for 11 or 13 cm IPG strips e Optional short plates for higher throughput of 7 cm IPG strips up to eight strips per run using divider plates Fig 6 SE 600 Ruby B First and second dimension electrophoresis with a flatbed system First dimension IEF using Multiphor II Electrophoresis System with Immobiline DryStrip Kit Rehydration in IPGbox Choice Factors e Can be used for both first and second dimension separations as well as for many other electrophoresis techniques e Versatile system for IEF with IPG strips from 7 to 24 cm e Note EPS 3501 XL Power Supply and MultiTemp III Thermostatic Circulator are required to supply power and cool the system respectively Fig 7 Multiphor II Electrophoresis System with Immobiline DryStrip Kit Second dimension SDS PAGE using Multiphor Il Electrophoresis System one 24 5 x 11 18 cm gel Choice Factors e Precast gels available ExcelGel SDS Homogeneous 12 5 24 5 x 11 cm and ExcelGel Gradient XL 12 14 24 5 x 18 cm e Relatively rapid 4 h or less for electrophoresis e High resolution All available IPG strip lengths can be used 80 6429 60AD 11 The experimental sequence for 2 D electrophoresis is 1 Sample preparation Proper sample preparation is absolutely essential for good 2 D results 2 Immobiline DryStrip gel rehydration Immobiline DryStr
41. groups in a protein from a sample being labeled For this reason the method has been called saturation labeling See Figure 62 for an overview of the workflow and Figure 63 for a diagram of the labeling process 114 80 6429 60 AD Pooled internal standard label with Cy3 Protein extract label with CyS a at Mix labeled extracts Image gel with Typhoon Variable Mode Imager Image analysis and data quantitation with DeCuder Differential Analysis Software Fig 62 Outline of the Ettan DIGE system workflow for saturation labeling 1 TCEP 37 C 1h dark Gos HS O 2 37 C 30 min dark FNAL 3 2x sample buffer H N gt 0 S IY N Cr oH Fig 63 Schematic of labeling reaction between CyDye DIGE Fluor saturation dye and the cysteine residues of a protein 80 6429 60 AD 115 6 3 Ettan DIGE system workflow The main steps in the Ettan DIGE system workflow are outlined in Figure 64 There are several key differences between standard 2 D electrophoresis and Ettan DIGE system experiments Failure to incorporate these changes into an Ettan DIGE system experiment will impact upon data quality See Table 41 for these differences y l Sample C 1 preparation 1st and 2nd a Determine dimension I protein separation I concentration Sample evaluation optimization I y Check lysate labeled with one z l dye on 1 D gel I Labeling l gt Check three aliquots of the interna
42. hood while wearing a disposable dust mask Follow all local safety rules and regulations for handling and disposal of materials Quick guide for finding information on gel casting for miniVE SE 260 and SE 600 Ruby electrophoresis systems To find gel casting information quickly refer to Table 29 for gel volumes required Table 30 for single percentage gel recipes and Table 31 for gradient gel recipes The instructions provided below for the preparation of vertical SDS polyacrylamide gels employ the Tris glycine system of Laemmli 78 84 80 6429 60 AD Protocol 1 Select the gel percentage See section 3 3 7 protocol instruction 1 Select the gel percentage Calculate the required casting solution volume The total volume of solution required depends on the gel size the gel thickness and the number of gels cast Table 29 gives volumes of gel solution required per gel Calculate the formulation of the gel solution The recipes given in Table 30 produce 100 ml of solution for a single percentage gel The recipes in Table 31 produce 50 ml each of light and heavy solution for a gradient gel These recipes can be scaled up or down depending on the volume required Prepare the gel solution Make up the gel solution without TEMED or ammonium persulfate Note An optional deaeration step may be performed at this point To do so make up the solution in a vacuum flask Add a small magnetic stirring bar Stopper the flask and apply a vac
43. in order to reduce heat damage to the stained proteins Cooling the gel prior to visualization can also help reduce degradation 2 If required pick any bands spots Note If manually picking bands spots it is advisable to place gels on a glass plate in order to reduce possible damage to the instrument surface Prolonged continuous exposure to a strong UV light source will degrade the Deep Purple Total Protein Stain signal with a half life in the region of 15 to 30 min Refer to the Deep Purple Total Protein Stain product instructions for additional information on re staining of gels alternative staining trays alternative imaging instruments use of Deep Purple Total Protein Stain with Ettan DIGE and cleaning of imaging instruments For cleaning and preparation of Bind Silane coated plates refer to appendix V The instructions accompanying Deep Purple Total Protein Stain also include a troubleshooting guide 140 80 6429 60 AD Appendix V Treating glass plates with Bind Silane Spot picking with Ettan Spot Picker or Spot Handling Workstation requires that gels are precast on backing e g Ettan DALT II Precast Gel 12 5 or immobilized on backing during casting Two different types of backing may be used Gel Bond PAGfilm or a glass plate treated with Bind Silane solution To scan a gel with fluorescently labeled proteins it is important that GelBond not be used for the gel backing GelBond is a plastic material and fluoresces intens
44. lift and lower the strip and slide it back and forth along the surface of the solution tilting the Strip Holder slightly as required to ensure complete and even wetting Finally lower the cathodic end of the Immobiline DryStrip gel into the channel making sure that the gel contacts the Strip Holder electrodes at each end The gel can be visually identified once the rehydration solution begins to enter the gel Be careful not to trap air bubbles under the Immobiline DryStrip gel 58 80 6429 60 AD 4 Apply Immobiline DryStrip Cover Fluid Apply Immobiline DryStrip Cover Fluid to minimize evaporation and thus prevent urea crystallization Pipette the fluid dropwise into one end of the Strip Holder until one half of the Immobiline DryStrip gel is covered Then pipette the fluid dropwise into the other end of the Strip Holder adding fluid until the entire gel is covered 5 Place the cover on the Strip Holder Pressure blocks on the underside of the cover ensure that the Immobiline DryStrip gel maintains good contact with the electrodes as the gel swells 6 Allow the Immobiline DryStrip gel to rehydrate Rehydration can proceed on the bench top or on the Ettan IPGphor 3 platform Ensure that the Strip Holder is on a level surface A minimum of 10 h is required for rehydration overnight is recommended The rehydration period can be programmed as the first step of an Ettan IPGphor 3 protocol This is especially convenient if temperature control durin
45. may be used 3 Pour off the stain solution and wash the gels by gentle rocking in wash solution C for 30 minutes This step should be increased to 45 min for 1 5 mm gels or if you experience high background fluorescence 4 Remove gels from wash solution and acidify by placing them in solution A and rock gently for 30 min This step may be repeated or extended up to overnight to reduce background staining If this step is prolonged to over night gels should be protected from light Prior to imaging gels should be rinsed 5 minutes in wash solution C The gel can be imaged at this stage Note If the gels swell during the staining process soak the gels in solution A for 30 min prior to analysis After imaging the gels can be stored at 4 C protected from light in gel 1 citric acid solution D For longterm storage more than 6 months add Deep Purple 1 200 dilution to the storage solution If stored gels should be rinsed 2 x 15 min in 15 ethanol solution C prior to imaging Acidifying in 15 ethanol 1 citric acid solution A for 15 minutes may be used to reduce background Visualization A Flat bed laser based fluorescence imaging systems 1 Ensure that the scanning bed of the laser is clean and free from smears and particles Follow recommended procedures provided with the instrument Note On the Typhoon scanner it has been shown that fluorescent contamination on the platen can be eliminated by wiping the surface with 10
46. most proteins to their fully random conformation with all ionizable groups exposed to solution Recently the use of thiourea in addition to urea has been found to further improve solubilization particularly of membrane proteins 10 16 55 57 Detergent A nonionic or zwitterionic detergent is always included in the sample solution to ensure complete sample solubilization and to prevent aggregation through hydrophobic interactions Originally either of two similar nonionic detergents NP 40 or Triton X 100 was used 1 2 Subsequent studies have demonstrated that the zwitterionic detergent CHAPS 2 4 is often more effective 58 for solubilizing a wide range of samples New zwitterionic detergents have been developed and are reported to improve the solubility of membrane proteins 59 60 When difficulties in achieving full sample solubilization are encountered the anionic detergent SDS can be used as a solubilizing agent SDS is a very effective protein solubilizer but because it is charged and forms complexes with proteins it cannot be used as the sole detergent for solubilizing samples for 2 D electrophoresis A widely used method for negating the interfering effect of SDS is dilution of the sample with a solution containing an excess of CHAPS Triton X 100 or NP 40 The final concentration of SDS should be 0 25 or lower and the ratio of the excess detergent to SDS should be at least 8 1 27 34 61 80 6429 60 AD 37 Reductant Red
47. not provided ce bath 1 5 ml capped microcentrifuge tubes microcentrifuge capable of at least 12 000 x g rehydration solution or EF sample solution for resuspension see next section vortex mixer Preliminary notes Procedure A is applicable for sample volumes of 1 100 ul containing 1 100 ug of protein For larger samples containing more than 100 ug of protein use procedure B Prior to starting the procedure chill the wash buffer to 20 C for at least 1 h A For sample volumes of 1 100 ul containing 1 100 ug of protein per sample Process the protein samples in 1 5 ml microcentrifuge tubes All steps should be carried out on ice unless otherwise specified 1 Transfer 1 100 ul of protein sample containing 1 100 ug protein into a 1 5 ml microcentrifuge tube 2 Add 300 ul of precipitant Mix well by vortexing or inversion Incubate the tube on ice 4 5 C for 15 min 3 Add 300 ul of co precipitant to the mixture of protein and precipitant Mix by vortexing briefly 4 Position the tubes in a microcentrifuge with cap hinges facing outward Centrifuge at maximum speed at least 12 000 x g for 5 min Remove the tubes from the microcentrifuge as soon as centrifugation has finished A small pellet should be visible mee Proceed rapidly to the next step to avoid resuspension or dispersion of the pellet 5 Remove as much of the supernatant as possible by decanting or careful pipetting Do not disturb the pellet 6 Carefully
48. number 17 1313 01 Acrylamide gel and other related electrophoresis reagents Citric acid Boric acid Sodium hydroxide High purity water double distilled RO or equivalent Ethanol Ammonium carbonate Acetonitrile Fr High purity water RO quality or better should be used as a diluent for Deep Purple Total Protein Stain and for preparing all gel processing solutions ET All reagents used should be of the highest quality available since any impurities can affect the background obtained on imaging PlusOne reagents from GE Healthcare are recommended Critical parameters Several critical parameters are important to the success of the Deep Purple Total Protein Stain protocol Review these parameters prior to beginning the procedure e Ensure that the containers used for gels are clean and do not contain any contaminants A wide variety of non metallic containers can be used with this stain including polypropylene polystyrene or Pyrex glass e Ensure that plates to be coated with Bind Silane are prepared to the highest standard e Use gloves that are not powdered Wash new gloves prior to handling plates containers or gels Any powder transferred to the gel may show up as speckles on images e During preparation of plates for gel casting employ methods that minimize generation of dust particles The use of any type of paper towel will generate particulate matter that will be visualized as speckles Plates should be clean
49. of low abundance proteins to bring the latter into detectable range This allows for improved resolution when an individual fraction is analyzed provides less crowded 2 D maps simplifies analysis and interpretation and increases the chances of discovering novel proteins of diagnostic or therapeutic interest 80 6429 60 AD 17 Precipitation of the proteins in the sample and removal of interfering substances are optional steps The decision to employ these steps depends on the nature of the sample and the experimental goal Precipitation procedures which are used both to concentrate the sample and to separate the proteins from potentially interfering substances are described in section 1 4 Sections 1 4 1 provides protocols for sample clean up using 2 D Clean Up Kit Section 1 5 discusses the effects that contaminants salts small ionic molecules albumin and IgG in human serum ionic detergents nucleic acids polysaccharides lipids and phenolic compounds might have on the 2 D result if they are not removed the section also discusses removal techniques that eliminate specific contaminants from the sample Protocols are provided for desalting using Mini Dialysis Kit section 1 5 1 removing undesirable nucleic acids using Nuclease Mix section 1 5 2 and eliminating problems associated with the presence of albumin and immunoglobulin G IgG from human plasma using Albumin and IgG Removal Kit section 1 5 3 In general it is advisable to keep
50. of the Multiphor II Electrophoresis System for 2 D electrophoresis is the fact that it can be used for both first dimension IEF and second dimension SDS PAGE Strip rehydration with or without samples is performed in the Immobiline DryStrip IPGbox After rehydration the Immobiline DryStrip gels are transferred to the electrophoresis unit for first dimension IEF 12 80 6429 60 AD EF The system is composed of the Multiphor II Electrophoresis System and Immobiline DryStrip Kit which also allows cup and paper bridge loading of the sample onto rehydrated Immobiline DryStrip gels using Immobiline DryStrip IPGbox This system accommodates up to 12 rehydrated Immobiline DryStrip gels of the same length for any one IEF protocol Power is supplied by the separate EPS 3501 XL power supply and temperature control by the separate MultiTemp Ill Thermostatic Circulator Table 2 shows the key operating differences between the Ettan IPGphor 3 Isoelectric Focusing System and Multiphor II Electrophoresis System for first dimension IEF Table 2 IEF system selection Maximum voltage Additional equipment required Time required for IEF Ettan IPGphor 3 10 000 V Multiphor II 3500 Vt Manifold plus Immobiline DryStrip IPGbox 2 36h Immobiline DryStrip IPGbox 2 72h Immobiline DryStrip Kit EPS 3501 XL Power Supply MultiTemp IlI Thermostatic Circulator Optimal focusing time varies widely depending on the Immobiline DryStrip gel length and p
51. per strip pl 7 125 11 200 13 250 18 340 24 450 Including sample if applied P von 36 Troubleshooting Immobiline DryStrip gel rehydration in IPGbox Symptom Possible cause Remedy Uneven or incomplete rehydration of strips Depending on the Immobiline DryStrip gel pH interval and the pH of the rehydration solution either the basic end or the acidic end will swell faster than the other The strip may not necessarily be of an even thickness following rehydration At the start of rehydration ensure that the rehydration solution is evenly distributed under the Immobiline DryStrip gel Move the gel strip back and forth to aid distribution The gel strip should float on the rehydration solution Unopened Immobiline DryStrip gel package was stored at or above room temperature for too long Store Immobiline DryStrip gels sealed at a temperature below 20 C Immobiline DryStrip gels were stored at or above room temperature for too long Do not allow dry Immobiline DryStrip gels o remain at room temperature for longer han 10 min as they will pick up moisture rom the air Incorrect volume of rehydration solution used Make sure the correct amount of rehydration solution according to Table 35 is added to he channel in the Immobiline DryStrip IPGbox Check calibration of pipettors Rehydration time is too short Rehydrate the Immobiline DryStrip gels or at least
52. same length can be accommodated for any one protocol An earlier product the Cup Loading Strip Holder is not included in the discussion that follows 2 1 Background to isoelectric focusing Isoelectric Focusing is an electrophoretic method that separates proteins according to their isoelectric points pl Proteins are amphoteric molecules they carry either positive negative or zero net charge depending on the pH of their surroundings Fig 16 The net charge of a protein is the sum of all the negative and positive charges of its amino acid side chains and amino and carboxyl termini The isoelectric point pl is the specific pH at which the net charge of the protein is zero Proteins are positively charged at pH values below their pl and negatively charged at pH values above their pl If the net charge of a protein is plotted versus the pH of its environment the resulting curve intersects the x axis at the isoelectric point Fig 16 Net Charge 4 COOH coo coc 43 2 NH3 NHS NH2 1 Isoelectric point pl COOH coo coo 0 11 pH 1 NH NHS NH2 2 pH lt pl pH pl pH gt pl 3 Fig 16 Plot of the net charge of a protein versus the pH of its environment The point of intersection of the curve at the x axis represents the isoelectric point of the protein 80 6429 60 AD 41 The presence of a pH gradient is critical to the IEF technique In a pH gradient and under the influence of an electric field a protein will move to
53. sample preparation as simple as possible A sample with low protein concentration and a high salt concentration for example could be desalted then concentrated by lyophilization or precipitated with TCA and ice cold acetone and resolubilized with rehydration solution In some instances the option of simply diluting the sample with rehydration solution may be sufficient If problems with protein concentration or interfering substances are otherwise insurmountable then precipitation or contaminant removal steps may be necessary 1 0 3 Additional aspects of sample preparation The composition of the sample solution is particularly critical for 2 D electrophoresis because solubilization treatments for the first dimension separation must not affect the protein pl or leave the sample in a highly conductive solution In general concentrated urea or combinations of urea and thiourea and one or more detergents are used Sample solution composition is discussed in section 1 6 Accurate quantitation of protein in samples prepared for electrophoresis can be difficult because many of the reagents used to prepare and solubilize samples for electrophoresis e g chaotropes carrier ampholytes detergents and reductants are incompatible with common protein assays Section 1 7 discusses this topic Section 1 7 1 provides a protocol for using 2 D Quant Kit to overcome this problem The above mentioned sample preparation kits from GE Healthcare simplify preparati
54. sandwich to the upper buffer chamber with the cams and release the gel sandwich from the caster 3 Fit the upper buffer chamber with the gel sandwiches onto the lower buffer chamber 4 Fill the upper buffer chamber with Laemmli SDS buffer 5 Attach or close the lid and connect the power leads to the power supply 6 Set the temperature control if desired 7 Stir the SE 600 Ruby lower tank buffer to maintain an even buffer temperature around the gels If using only one gel in SE 600 Ruby and SE 260 units the second side of the unit will need to be blocked with a buffer dam assembly or two glass plates clamped together no spacers to prevent current leakage For detailed information please consult the respective instrument user manuals 3 4 7 Electrophoresis conditions Table 33 lists the recommended conditions for miniVE SE 260 and SE 600 Ruby Electrophoresis is performed at constant current in two steps During the initial migration and stacking period Step 1 the current is approximately half of the value required for the separation Step 2 as can be seen from Table 33 Stop electrophoresis when the dye front is approximately 1 mm from the bottom of the gel For these vertical systems cooling is optional However temperature control improves gel to gel reproducibility especially if the ambient temperature of the laboratory fluctuates significantly For best results gels should be run at 25 C 80 6429 60 AD 87 Afte
55. standard should be incorporated within each gel The ideal internal standard comprises pooled aliquots from all the biological samples within the experiment The internal standard is labeled with one CyDye DIGE Fluor minimal dye e g Cy2 and is run on every gel together with experimental samples labeled with Cy3 or Cy5 CyDye DIGE Fluor minimal dyes Table 42 This ensures that every spot on every gel is represented within the common internal standard Each protein spot in a sample can therefore be compared with its representative within the internal standard to generate a ratio of relative expression Fig 65 Experimental design for using the two saturation dyes from the CyDye DIGE Fluor Labeling Kit for Scarce Samples and Preparative Gel Labeling is simple one dye is selected to label the internal standard e g Cy3 and the other to label the individual samples in the experiment 80 6429 60 AD 117 Gel 1 Gel 2 Sample 2 Cy5 Sample 4 Cy5 Sample 1 Cy3 Sample 3 Cy3 e e J e ee eee Protein N sample 1 Protein N standard gel 1 Protein N sample 3 Protein N standard gel 2 Protein N sample 2 Protein N standard gel 1 Protein N sample 4 Protein N standard gel 2 Fig 65 Quantitation of protein abundance using co detection algorithms From each gel three scan images are generated Cy2 for the internal standard and Cy3 or Cy5 for test samples The protein abundance of
56. the position in the gradient where its net charge is zero A protein with a net positive charge will migrate toward the cathode becoming progressively less positively charged as it moves through the pH gradient until it reaches its pl A protein with a net negative charge will migrate toward the anode becoming less negatively charged until it also reaches zero net charge If a protein should diffuse away from its pl it immediately gains charge and migrates back This is the focusing effect of IEF which concentrates proteins at their pls and allows proteins to be separated on the basis of very small charge differences The resolution is determined by the slope of the pH gradient and the electric field strength IEF is therefore performed at high voltages typically in excess of 1000 V When the proteins have reached their final positions in the pH gradient there is very little ionic movement in the system resulting in a very low final current typically in the microamp range IEF of a given sample in a given electrophoresis system is generally performed for a constant number of Volt hours Volt hour Vh being the integral of the volts applied over the separation time IEF performed under denaturing conditions gives the highest resolution and the sharpest results Complete denaturation and solubilization is achieved with a mixture of urea detergent and reductant ensuring that each protein is present in only one conformation with no aggregation th
57. undesirable nucleic acids from samples using Nuclease MIX 34 1 5 3 Simultaneous DNA RNA and protein isolation from undivided scarce samples 34 1 5 4 Using Albumin and IgG Removal Kit to improve 2 D electrophoresis Of NUMAN SEUN ccccsssssssssssssssssssssesesseseeeeesessssssssssssssuussssssesesseeeeeeeseee 34 1 6 Composition of sample preparation Solution eccccceccccccccsssssssssssssssssssssssesesseeceeessessssssssssssuueessseses 37 1 6 1 Components of sample Preparation SOLUTIONS ne ececcccsccssscccccscssssesssssssussssssessseseeeeeessee 37 1 6 2 Examples of sample preparation SOLUtIONS eesssssssssscccececeessssssssnsnnusesesesseseceeeeeeeetes 38 1 7 QUENTITALING protein SCMPISS s ceccic czccesscd cisscevestscasuaccesetsvanczssdecsnieccsncsecssbasadaaadia arbatacceeassccnaddsscentesttaasetateaes 38 1 7 1 Protein determination USING 2 D Quant Kit neesssssssscccccecsesssssssnnssnusssssesesseeeceeeeeeeete 39 DB SGMMPIG TOGA Sien aee E e E AE E tts RESA 40 2 First dimension isoelectric focusing IEF e sesssssressssesessesssesssressrersseessrerssseesreesseeessreesneeessressrerssee 41 ZO OM STOW Ere aeee aa aaa e 41 2 1 Background to isoelectric FOCUSING weeeeesessssssssssscsccccscsssssssssssnsunusssssesseseeeeeeceeessssssssussssimussseseceeseeeeeseeseee 41 2 2 IMMOBlNneDryStip gels neiniau i 43 2 2 1 Choosing strip length eeeeeeesccccccccccssssesssssssssssnussuusssseeseeseseeeeeeecssssssssssnssuusssssssseeseseeeeseeeeesssssansnsananee
58. 0 For further information on compatible reagents for labeling please refer to appendix E 2 of the Ettan DIGE user manual Protocol for preparing protein from cell cultures and then labeling with CyDye DIGE Fluor minimal dyes A Washing cells If using a cell culture wash cells to remove any growth media or reagents that might affect the CyDye DIGE Fluor minimal dye labeling process Check that the cell wash buffer does not contain any primary amines or thiols that may interfere with the downstream labeling process DIGE cell washing buffer contains 10 mM Tris pH 8 0 5 mM magnesium acetate DIGE cell lysis buffer contains 30 mM Tris 2 M thiourea 7 M urea 4 CHAPS w v at pH 8 5 B Lysing cells in lysis buffer 1 Resuspend the washed cell pellet in 1 ml of DIGE cell lysis buffer at pH 8 5 and leave on ice for 10 min approximately 4 x 10 E coli cells will yield 5 10 mg protein 2 Lyse cells see section 1 1 on ice until solution becomes less cloudy 3 Centrifuge to pellet cell debris 80 6429 60 AD 119 4 Transfer supernatant into a tube and if necessary adjust to pH 8 5 5 Determine protein concentration For best results the sample concentration should be 1 10 mg ml For further information relating to sample preparation please refer to the Ettan DIGE user manual If working with a sample in an unknown buffer the sample should be precipitated and resuspended in an Ettan DIGE system compatible buffer 2 D Cle
59. 0 cm in 40 s at 300 dpi e Typhoon 9400 Variable Mode Imager has red green and blue excitation wavelengths and a wide choice of emission filters that enable imaging of a variety of fluorphores Typhoon series imagers can be used for high performance four color automated fluorescence detection making them ideal for use with the three dye system employed in 2 D DIGE analysis with Ettan DIGE system In addition Typhoon 9400 series imagers perform storage phosphor imaging and chemiluminescence imaging Comprehensive information on fluorescence imaging can be found in the GE Healthcare handbook Fluorescence imaging principles and methods see additional reading and reference material e ImageMaster 2D Platinum is a high throughput 2 D imaging software for almost parameter free spot detection No manual spot editing is required resulting in maximum reproducibility of evaluation results Matching is based on spot features rather than simply spot positions A wide selection of statistical tools enables the user to extract the relevant information in a minimum of time with maximum confidence e DeCyder 2 D Differential Analysis Software has been specifically developed as a key element of the Ettan DIGE system and is described further in chapter 6 In addition to these products Personal Densitometer SI is also available Personal Densitometer is a highly sensitive laser based transmission densitometer with a linear range of 0 1 3 5 OD that
60. 1 NL 17 6003 77 pH 3 5 4 5 17 6002 38 pH 4 7 17 6002 46 pH 6 9 17 6002 47 pH 3 7 NL 17 6002 43 pH 6 2 7 5 17 6003 67 pH 7 11 NL 17 6003 72 Equilibration Tube Set for up to 12 pk 80 6467 79 24 cm IPG strips 152 80 6429 60 AD Product Quantity Code No DeStreak rehydration reagents DeStreak Rehydration Solution 5x3ml 17 6003 19 DeStreak Reagent 1ml 17 6003 18 IPG Buffer 1 ml PH 3 5 5 0 17 6002 02 PH 5 5 6 7 17 6002 06 pH 4 7 17 6000 86 pH 6 11 17 6001 78 pH 3 10 17 6000 87 pH 3 10 NL 17 6000 88 pH 7 11 NL 17 6004 39 pH 3 11 NL 17 6004 40 Pharmalyte 25 ml pH 3 10 17 0456 01 pH 5 8 17 0453 01 pH 8 10 5 17 0455 01 Second dimension products and accessories Mini Vertical units and accessories miniVE complete includes 3 rectangular glass plates 3 notched plates 80 6418 77 2 gel modules lid lower buffer chamber 2 each 1 0 mm thick 10 well combs and 1 0 mm thick spacer sets glass plate size 10 x 10 5 cm Spacer 1 0 mm 2 pk 80 6150 11 Spacer 1 5mm 2 pk 80 6150 30 SE 250 Mini Vertical Unit 80 6147 45 complete for 2 slab gels gel format 10 x 8 cm SE 260 Mini II Vertical Unit 80 6149 35 complete for 2 slab gels gel format 10 x 10 5 cm SE 235 Mighty Small 4 Gel Caster complete 80 6146 12 SE 245 Mighty Small Dual Gel Caster 80 6146 50 Wonder Wedge plate separation tool 80 6127 88 SE 600 Ruby Vertical Electrophoresis System and accessories SE 600 Ruby Dual Cooled Vertical Gel Unit for 80 6479 57 up t
61. 10 SDS see solution J above 0 1 10 ml Double distilled water to1l Store at 4 C M 1x Laemmli SDS electrophoresis buffer 25 mM Tris base 192 mM glycine 0 1 SDS 10 Final concentration Amount Tris base FW 121 1 25mM 30 3 g Glycine FW 75 07 192 mM 144 0 g SDS FW 288 38 0 1 w v 10 0 g Double distilled water to 101 The pH of this solution should not be adjusted This solution can be prepared by diluting one volume of 10x Laemmli SDS buffer solution F with nine volumes of double distilled water Store at room temperature N Agarose sealing solution 25 mM Tris base 192 mM glycine 0 1 SDS 0 5 agarose 0 002 bromophenol blue 100 ml Final concentration Amount Laemmli SDS electrophoresis buffer see solution M 100 ml Agarose NA or M 0 5 0 5g 1 Bromophenol blue stock solution 0 002 w v 200 ul Add all ingredients into a 500 ml Erlenmeyer flask Swirl to disperse Heat in a microwave oven on low or on a heating stirrer until the agarose is completely dissolved Do not allow the solution to boil over Dispense 1 5 ml aliquots into screw cap tubes and store at room temperature 132 80 6429 60 AD Appendix Il Optimized silver staining of large format DALT gels and DALT 12 5 precast gels using PlusOne Silver Staining Kit Protein Prepare staining reagents 250 ml per gel according to the PlusOne Silver Staining Kit Protein instructions with the foll
62. 15 min For each original volume of sample add three volumes of co precipitant to the mixture of protein and precipitant Mix by vortexing briefly Position the tubes in a microcentrifuge with the cap hinges facing outward Centrifuge at 8000 x g for 10 min Remove the tubes from the microcentrifuge as soon as centrifugation has finished A pellet should be visible Er Proceed rapidly to the next step to avoid resuspension or diffusion of the pellet Remove as much of the supernatant as possible by decanting or careful pipetting Do not disturb the pellet Carefully position the tubes in the microcentrifuge with the cap hinges and pellets facing outward Centrifuge the tubes for at least 1 min to bring any remaining liquid to the bottom of the tubes Use a pipette to remove the remaining supernatant There should be no visible liquid remaining in the tubes To each tube add three fold to four fold more co precipitant than the size of the pellet Carefully reposition the tubes in the microcentrifuge with the cap hinges facing outward Centrifuge for 5 min Use a pipette to remove the supernatant Pipette enough distilled or deionized water on top of each pellet to cover the pellet Vortex each tube for several seconds The pellets should disperse but not dissolve in the water 0 Add 1 ml of wash buffer prechilled for at least 1 h at 20 C to each tube For an initial sample volume of 0 1 0 3 ml add 1 ml of wash buffer However the vol
63. 2 pk 80 6474 63 Ettan DALT Precast Gel Cassette 80 6466 65 Ettan DALT Gel Casting Cassette 1 0 mm hinged cassette 80 6466 84 Ettan DALT Gel Casting Cassette 1 5 mm hinged cassette 80 6488 69 Ettan DALT Blank Cassette Insert 80 6467 03 Roller for precast gels 80 1106 79 Wonder Wedge plate separation tool for lab cast gels 80 6127 88 Ettan DALT Separator Sheets 0 5 mm 16 pk 80 6467 41 Ettan DALT Filler Sheets 1 0 mm 6 pk 80 6467 60 Ettan DALT Cassette Rack 2 pk 80 6467 98 Ettan DALT Glass Plate Set including spacers 1 set of 2 pcs 80 6475 39 standard glass plates for spot picking Ettan DALT Low Fluorescence Glass Plate Set including spacers 1 set of 2 pcs 80 6475 58 Equilibration Tube Set 12 80 6467 79 Staining Tray Set 80 6468 17 Ettan DALTsix Gradient Maker 80 6487 36 DALT Gradient Maker with peristaltic pump 115 V 80 6067 65 DALT Gradient Maker with peristaltic pump 230 V 80 6067 84 DIGE gels and buffer kit DIGE gel 3 28 9374 51 DIGE Buffer Kit 28 9374 52 2 x 125 ml anode buffer 4x 125 ml cathode buffer 12 tubes agarose sealing solution Ettan DALT precast gels and buffer kit DALT Gel 12 5 6 pk 17 6002 36 DALT Buffer Kit 17 6002 50 Gradient makers SG15 Gradient Maker 15 ml total volume 80 6197 61 SG 30 Gradient Maker 30 ml total volume 80 6197 80 SG 50 Gradient Maker 50 ml total volume 80 6197 99 SG 100 Gradient Maker 100 ml total volume 80 6196 09 SG 500 Gradient Maker 500 ml total volume 80 6198 18 Multiphor II Electr
64. 5 nmol RPKO272 CyDye DIGE Fluor Cy3 minimal dye 5 nmol 25 8010 83 CyDye DIGE Fluor Cy3 minimal dye 10 nmol 25 8008 61 CyDye DIGE Fluor Cy3 minimal dye 25 nmol RPK0273 CyDye DIGE Fluor Cy5 minimal dye 5 nmol 25 8010 85 CyDye DIGE Fluor Cy5 minimal dye 10 nmol 25 8008 62 CyDye DIGE Fluor Cy5 minimal dye 25 nmol RPK0275 CyDye DIGE Fluor Labeling Kit for Scarce Samples 25 8009 83 for a minimum of 12 labeling reactions CyDye DIGE Fluor Labeling Kit for Scarce Samples plus Preparative Gel Labeling 25 8009 84 for minimum of 12 labeling reactions and 1 prep gel CyDye DIGE Fluor Preparative Gel Labeling Kit for Scarce Samples 28 9366 83 CyDye DIGE Kit 2 nmol 28 9345 30 DIGE Trial pack CyDye DIGE Kit 2 nmol DeCyder 1 month trial license 28 9373 73 Ettan DIGE Gel Alignment Guides for SE600 80 6496 29 Ettan DIGE Gel Alignment Guides for Ettan DALT 80 6496 10 Imaging systems Typhoon Variable Mode Imagers Typhoon FLA 9000 1 28 9558 08 Typhoon FLA 7000 1 28 9558 09 Typhoon 9400 63 0055 78 Typhoon 9410 1 63 0055 80 Typhoon Trio 1 63 0055 87 Typhoon Trio 1 63 0055 89 ImageQuant imagers ImageQuant LAS 4000 1 28 9558 10 ImageQuant LAS 4010 1 28 9558 11 ImageQuant LAS 4000 mini 1 28 9558 13 Other imaging systems ImageScanner III 1 28 9076 07 Storm 820 and ImageQuant TL 1 28 9328 12 Storm 845 and ImageQuant TL 1 28 9326 41 Storm 865 and ImageQuant TL 1 28 9327 91 80 6429 60 AD 157 Product Quantity Code No Analysis s
65. 7 Pressing out air pockets between gel and glass plate Fig 38 Closing the DALT Precast Gel Cassette 3 3 3 Equilibrating Immobiline DryStrip gels Refer to section 3 1 2 The equilibration procedure is the same whether applying the strip to precast or lab cast gels 3 3 4 Applying equilibrated Immobiline DryStrip gels to SDS gels Both types of DALT gel cassettes those for precast and for lab cast gels have a longer glass plate The cassette should be laid on the bench with the longer glass plate down and the protruding edge oriented toward the operator Fig 39 Protocol 1 Position the Immobiline DryStrip gel Dip the equilibrated Immobiline DryStrip gel see section 3 1 2 in the SDS electrophoresis buffer see appendix solution M to lubricate it If using the DALT Gel 12 5 the diluted cathode buffer can be used to lubricate the strip Place the strip with the acidic end to the left gel surface up onto the protruding edge of the longer glass plate Fig 39 If using a system other than DALTtwelve or DALTsix position the Immobiline DryStrip gel between the plates on the surface of the second dimension gel with the plastic backing against one of the glass plates 2 Ensure Immobiline DryStrip gel has good contact With a thin plastic ruler gently push the Immobiline DryStrip gel down so that the entire lower edge of the Immobiline DryStrip gel is in contact with the top surface of the slab gel Fig 40 Ensure that no air
66. 74 3 3 2 Inserting DALT Gel 12 5 into DALT Precast Gel Cassette wccccccccccccsssessesssssssssnnmneses 76 3 33 Equilibrating IMMobiline DryStrip Gels ecceecccccccccsssssssssssssssssessessessesceceeceeesssssssssssssnueueseeeses 77 3 3 4 Applying equilibrated Immobiline DryStrip gels to SDS Gel eeeeeccesssssssssssssssssssneeesses 77 3 3 5 Inserting gels into Ettan DALT electrophoresis Units oe ecssscsssscccceccecceessssssssssnnmnneeees 78 3 3 6 Electrophoresis conditions with precast gels for both Ettan DALTsix ANd Ettan DALT WElV 80 3 3 7 Preparing lab cast gels 3 3 8 Preparing Ettan DALT electrophoresis units for electrophoresis 3 4 sing lab cast GEIS ssssesssssssessssssesesesessecccececcesssssssssssnsnsnussesseseeeeeeees 83 3 3 9 Equilibrating Immobiline DryStrip gels With lab cast gels ccecceecccccssssssssssssssssseessee 83 3 3 10 Applying Immobiline DryStrip gels to lab Cast gels oc sssssssccccecscessssssssssssneeseeses 83 3 3 11 Inserting lab cast gels into Ettan DALT electrophoresis UNItS nc 83 3 3 12 Electrophoresis conditions With lab cast GEIS essssssesssessssssesssseecccccessessssssssssssssuneeeeseesee 83 5 35 15 MOUDIGSMOOUMG sian cacs kececseccohs ER A a AA 83 Electrophoresis using other vertical electrophoresis SYStOM ccsssssssssssssssessssssesecssseseceseeeeee 84 3 4 1 Preparing caster and gel sandwich for miniVE SE 260 and SE 600 Ruby electrophoresis SYStOMS cccccccssssscsscccssssssssssssnussessssess
67. DryStrip gels Larger quantities of protein require more time to focus Focusing for substantially longer than recommended will cause horizontal streaking and loss of proteins This phenomenon is called over focusing Therefore focusing time should be reduced to the minimum necessary see chapter 7 Troubleshooting 4 1 6 Protocol examples The protocols in Table 37 are suitable for first dimension isoelectric focusing of protein samples in typical analytical quantities Table 16 with IPG Buffer concentrations of 0 5 to 2 in the rehydration solution The optimal focusing time will vary with the nature of the sample the amount of protein and how the sample is applied er For higher protein loads up to 1 mg or more the final focusing step of each protocol can be extended by an additional 20 of the total recommended Volt hours if necessary Sample application onto pH 6 11 pH 6 9 and pH 7 11 NL Immobiline DryStrip gels by rehydration loading is less likely to give high quality 2 D results and should be avoided Samples should be applied using cup loading at the acidic end of the Immobiline DryStrip gel coupled with use of DeStreak Reagent 96 80 6429 60 AD 4 1 7 Running a Multiphor II protocol Ensure that the electrodes in the Immobiline DryStrip tray are connected and place the lid on the Multiphor II unit Connect the leads on the lid to the power supply Ensure that the current check on the EPS 3501 XL Power Supply is switch
68. Ensure instantaneous mixing by introducing the reagent as rapidly as possible Incubate at room temperature for 15 20 min Read the absorbance at 480 nm for each sample and standard using a spectrophotometer such as Ultrospec1100 pro UV Visible Spectrophotometer Generate standard curve by plotting the absorbance of the standards against the quantity of protein Estimate protein concentration of samples by comparison to the standard curve 1 8 Sample loads The optimal quantity of protein to load for electrophoresis varies widely depending on factors such as sample complexity the length and pH range of the Immobiline DryStrip gel and the method of visualizing the 2 D separation General sample load guidelines for different staining techniques are given in chapter 2 Table 16 40 80 6429 60 AD 2 First dimension isoelectric focusing IEF 2 0 Overview GE Healthcare offers two flatbed electrophoresis systems for first dimension separation using isoelectric focusing IEF Ettan IPGphor 3 Isoelectric Focusing System and Multiphor II Electrophoresis System This chapter provides information on Ettan IPGphor 3 information specific to Multiphor II is covered in chapter 4 Ettan IPGphor 3 Isoelectric Focusing System comprises Immobiline DryStrip gel strips which contain an immobilized pH gradient IPG and are commonly referred to as IPG strips two accessory options for holding the strips in place the Manifold and fixed length Strip Ho
69. F should be solubilized in water while samples for denaturing IEF for 2 D work should be solubilized in a solution containing urea reductant and nonionic detergent See sections 1 6 1 and 1 6 2 for details CP Handle dialysis tubes and caps with gloves v Dialysis tubes are supplied in 0 05 w v sodium azide and require rinsing before use 1 Rinse dialysis tube and cap with distilled or deionized water Fig 11A Keep cap covered with water until needed Do not allow cap with dialysis membrane to dry out 2 Remove cap from water Remove excess water with a micropipette Ensure that the cap is tightly sealed 3 Add sample to dialysis tube and replace dialysis cap Fig 11B For 250 ul dialysis tubes use 10 250 ul of sample For 2 ml dialysis tubes use 200 ul 2 ml of sample 4 Invert dialysis tube ensuring that entire sample rests on dialysis membrane If the sample is viscous and does not initially rest on the membrane the dialysis tube can be centrifuged in the inverted position at 10 100 x g for no more than 6 s W Spinning longer or faster may rupture the membrane 5 Secure dialysis tube to one of the floats provided Place dialysis tube and float assembly cap end down in a beaker of the solution to be dialyzed against e g water or 1 glycine for native IEF or sample buffer containing urea reducing agent and nonionic detergent for denaturing IEF see sections 1 6 1 and 1 6 2 for details Check that the dialysis membran
70. Fluor Cy5 minimal dye This setup avoids repeatedly linking the same two treatment types on multiple gels For further information relating to experimental design please refer to the Ettan DIGE user manual 6 3 2 Sample preparation for Ettan DIGE system applications CyDye DIGE Fluor minimal dyes used for protein labeling in Ettan DIGE system applications form a peptide linkage between the fluor and lysine residues within the protein Components such as primary amines e g ampholytes will compete with the proteins for fluor binding Thiols e g DTT also cause a reduction in labeling efficiency The result will be fewer fluor labeled proteins which may affect the data after scanning and spot detection To achieve optimal labeling such components should be omitted from both the lysis and sample buffers and are only added to the sample after labeling The labeling reaction with CyDye DIGE Fluor minimal dyes is most efficient at pH 8 5 Below pH 8 0 reactivity of the label is reduced above pH 9 0 increased non specific binding to thiol groups is promoted and the NHS ester may become inactivated due to hydrolysis Lysis and sample solutions should be buffered using NaOH to pH 8 5 CyDye DIGE Fluor saturation dyes included in the labeling kits for scarce samples and preparative gels covalently bind to the thiol group of cysteine residues via a thioether linkage The labeling reaction with CyDye DIGE Fluor saturation dyes is most efficient at pH 8
71. GE Healthcare 2 D Electrophoresis Principles and Methods Handbooks from GE Healthcare z A pl 2 i P 7 y Sas p y a a9 4 separanion 0 Puntfying ie vont panes ne halengi ee 7 ti o B a 9 8 o B z Q 8 GST Gene Fusion System Handbook 18 1157 58 Affinity Chromatography Principles and Methods 18 1022 29 Antibody Purification Handbook 18 1037 46 lon Exchange Chromatography and Chromatofocusing Principles and Methods 11 0004 21 Cell Separation Media Methodology and Applications 18 1115 69 Purifying Challenging Proteins Principles and Methods 28 9095 31 Isolation of mononuclear cells Methodology and Applications 18 1152 69 High throughput Process Development with PreDictor Plates Principles and Methods 28 9403 58 f i Ay POPC issketlon ut fmonenucteor cej een MTD pir 4 8 g gt 9 Gel Filtration Principles and Methods 18 1022 18 Recombinant Protein Purification Handbook Principles and Methods 18 1142 75 Protein Purification Handbook 18 1132 29 Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods 11 0012 69 2 D Electrophoresis using immobilized pH gradients Principles and Methods 80 6429 60 Microcarrier Cell Culture Principles and Methods 18 1140 62 Nucleic Acid Sample Preparation for Downstream Analyses Principles and Methods 28 9624 00 2 D Electrophoresis Principles and Meth
72. H range and the nature of the sample Similar separations can generally be performed at least two fold faster with the Ettan IPGphor 3 Isoelectric Focusing System than with the Multiphor II Electrophoresis System t Higher voltages are not recommended for safety reasons Guidelines for the selection of sample application methods for Ettan IPGphor 3 Isoelectric Focusing System and Multiphor II Electrophoresis System and can be found in sections 2 4 and 4 1 3 4 1 4 respectively Selecting a second dimension system The second dimension separation may be performed in a vertical or flatbed system Table 3 lists the appropriate second dimension system for a given gel size and Immobiline DryStrip gel length Further considerations are discussed below For a more complete discussion of the relative merits of vertical compared with flatbed second dimension systems see reference 8 Table 3 Selection of second dimension electrophoresis system with suggested Immobiline DryStrip and precast slab gels Approx gel size Number of Gel thickness IPG strip length Total separation time w x l cm gels mm cm h m Vertical Ettan DALTsix 26 x 20 1 6 1 1 5 18 24 4 00 6 30 Ettan DALTtwelve 26 x 20 1 12 115 18 24 5 00 7 00 miniVE or SE 260 8x95 1 2 1 15 7 1 30 SE 600 Ruby 14x 16 1 4 115 11 3 00 5 00 16 x 16t 1 4 L15 13 3 00 5 00 16 x 88 1 4 1 15 13 3 00 4 00 Flatbed Multiphor II ExcelGel 2 D Homogeneous 12 5 24 5 11 1 0 5
73. IPGphor Manifold and 10 kV set current limit to 75 pA per strip and follow step 1 2 3b and 4b Using IPGphor Regular Strip Holder or Cup Loading Strip Holder with the 18 and 24 cm strips the maximum allowed voltage is 8000 V and current 50 pA per strip Follow step 1 2 3a 4a pH Step Voltage Voltage Time Volt hours intervals mode v h min kVh 3 10 1 Step and Hold 500 1 00 8 00 0 5 3 11 NL 2 Gradient 1000 1 00 0 8 6 11 3a Gradientt 8000 3 00 13 5 4a Step and Holdt 8000 0 46 1 30 6 2 12 2 3b Gradient 10000 3 00 16 5 4b Step and Hold 10000 0 20 0 55 3 2 9 2 Total 21 0 27 0 3 10 NL 1 Step and Hold 500 1 00 8 00 0 5 4 7 2 Gradient 1000 1 00 0 8 3 5 6 NL 3a Gradient 8000 3 00 13 5 4a Step and Holdt 8000 1 30 2 40 12 2 21 2 3b Gradient 10000 3 00 16 5 4b Step and Hold 10000 0 55 1 50 9 2 18 2 Total 27 0 36 0 6 9 1 Step and Hold 500 1 00 8 00 0 5 7 11 NL 2 Gradient 1000 1 00 0 8 3a Gradient 8000 3 00 13 5 4a Step and Holdt 8000 3 10 4 30 25 2 35 2 3b Gradient 10000 3 00 16 5 4b Step and Holdt 10000 2 15 3 15 22 2 32 2 Total 40 0 50 0 5 3 6 5 1 Step and Hold 500 2 00 3 00 1 0 6 2 7 5 2 Gradient 1000 2 00 15 3a Gradientt 8000 3 00 13 5 4a Step and Holdt 8000 6 45 8 40 54 0 69 0 3b Gradient 10000 3 00 16 5 4b Step and Hold 10000 5 05 6 35 51 0 66 0 Total 70 0 85 0 When a more convenient overnight run of 15 to 17 his desired the time in step 1 can be extended up to recommended value in brac
74. Immobiline DryStrip gel by rehydration the sample volumes shown in Table 10 should be used according to the length of the Immobiline DryStrip gel Table 10 Sample volumes for different Immobiline DryStrip gel lengths Immobiline DryStrip gel length cm Sample volume applied ul 7 125 1L 200 13 250 18 340 24 450 The optimal quantity of protein to load varies widely depending on factors such as sample complexity the length and pH range of the Immobiline DryStrip gel and the method of visualizing the 2 D gel separation General guidelines are given in chapter 2 The protein concentration of the sample is best determined using the 2 D Quant Kit which can accurately quantitate protein in the presence of detergents reductants and other reagents used in sample preparation See section 1 7 for details 80 6429 60 AD 29 1 5 Other methods for removing contaminants The first dimension IEF step of 2 D electrophoresis is particularly sensitive to low molecular weight ionic impurities Non protein impurities in the sample can interfere with separation and subsequent visualization of the 2 D gel result so sample preparation may require steps to rid the sample of these substances Table 11 lists contaminants that affect 2 D results and techniques for their removal Reference 9 provides further discussion on the removal of interfering substances Mini Dialysis Kit Albumin and IgG Removal Kit and Nuclease Mix may be used to remov
75. Immobiline DryStrip gel to the lab cast gel follow the procedure as described in section 3 3 4 3 3 11 Inserting lab cast gels into Ettan DALT electrophoresis units To insert lab cast gels into Ettan DALT electrophoresis units follow the procedure described in section 3 3 5 3 3 12 Electrophoresis conditions with lab cast gels Follow the procedure described in section 3 3 6 3 3 13 Troubleshooting See section 3 5 80 6429 60 AD 83 3 4 Electrophoresis using other vertical electrophoresis systems Several other electrophoresis units work well for second dimension separation Choice is to a large degree dependent on the length of the Immobiline DryStrip used in the first dimension Two systems miniVE and SE 260 are ideal for running up to two second dimension gels with 7 cm Immobiline DryStrip gels Spacers 1 0 and 1 5 mm are available as well as two plate lengths 8 or 10 5 cm SE 600 Ruby units can be used to cast and run up to four gels 16 cm in length Divider plates allow two gels to be cast and run together on each side of the gel tank The width of the gels can either be 14 or 16 cm depending on the width of the spacers chosen which allows SE 600 Ruby to accommodate either 11 or 13 cm Immobiline DryStrip gels respectively Several gel casters are available including a 10 gel caster Low fluorescent glass plates are also available for use in SE 600 Ruby SE 600 Ruby requires an external power supply such as the EPS 601
76. Immobiline DryStrip pH 4 7 Ettan IPGphor Isoelectric Focusing System 17 5 kVh Second dimension SDS PAGE 12 5 SE 260 8 x 9 cm gel Stain Silver Staining Kit Protein 26 80 6429 60 AD Table 9 Effect of sample preparation on the number of protein spots detected in 2 D electrophoresis gels Sample preparation Number of silver stained spots Protein extracted with urea buffert 726 Protein extracted with 1 Triton X 100 and precipitated with three volumes of acetone 758 Protein extracted with 1 Triton X 100 and purified using 2 D Clean Up Kit 801 Protein spots were detected using ImageMaster 2D Elite software t 9 8 M urea 2 CHAPS 0 5 IPG Buffer pH 3 10 65 mM DTT The 2 D Clean Up Kit procedure uses a combination of a unique precipitant and co precipitant to quantitatively precipitate the sample proteins while leaving interfering substances behind in the solution The proteins are pelleted by centrifugation and the precipitate is washed to further remove non protein contaminants The mixture is centrifuged again and the resultant pellet can be easily resuspended into a 2 D sample solution of choice compatible with first dimension IEF The kit contains sufficient reagents to process 50 samples of up to 100 ul each The procedure can be scaled up for larger volumes or more dilute samples Protocol 2 D Clean Up Kit Reagents supplied Precipitant co precipitant wash buffer wash additive Required but
77. Make sure all the slots in the electrophoresis unit are filled with either a gel or a blank cassette Acrylamide solution is too old Prepare fresh monomer stock solution Dye front curves up Gel temperature is not uniform Regulate gel temperature using a thermostatic smiles at the edges circulator Use the maximum possible volume of buffer in the lower reservoir Current or power too high Limit current or power to values suggested in Table 23 Dye front curves Gel is poorly polymerized near Degas the gel solution or increase the amount of down frowns the spacers ammonium persulfate and TEMED by 50 Improper instrument assembly Ensure that the gasket is not pinched SE 600 Ruby Leakage of upper reservoir Ensure that an adequate level of buffer is in the upper reservoir continues on following page 88 80 6429 60 AD Table 34 Troubleshooting vertical second dimension SDS PAGE continued Symptom Possible cause Remedy Second dimension All of the slots in the sealing assembly are Ensure that all slots in the electrophoresis unit separation proceeds not occupied by either gel cassettes or are occupied slowly with high blank cassettes current Anodic buffer has mixed with cathodic Do not pour more than the suggested volume 7 5 I buffer from overfilling of either the into the lower reservoir cathodic reservoir or the anodic reservoir Ensure that the level of the anode buffer does not Ettan DALT syst
78. able cassette and run vertically in the Ettan DALT systems If fluorescent staining labeling techniques will be used do not run gels cast on plastic backing as it can pose a problem of high background with some dyes during subsequent analysis 3 3 1 Preparing Ettan DALT system for electrophoresis using precast gels Protocols for use of Ettan DALTsix and Ettan DALTtwelve differ in two main areas preparing the system and inserting the gel into the unit Thus where appropriate separate protocols are provided for the different systems The first instance follows Protocol Preparing Ettan DALTsix For detailed instructions for using Ettan DALTsix system please refer to the Ettan DALTsix user manual Preliminary steps Place the unit close to a sink for easy rinsing and draining Connect the tubing leading to and from the heat exchanger to a thermostatic temperature controller such as MultiTemp IIl Do not connect the heat exchanger to a water tap or any other coolant supply that does not have pressure regulation Position an EPS 601 Power Supply conveniently close to the electrophoresis unit 1 Prepare anode and cathode buffers stocks included in the DALT Buffer Kit Dilute half of the 100x anode lower buffer by adding 37 5 ml to 4 5 of water Dilute one bottle of 10x cathode upper buffer to 2x buffer to a final volume of 1 2 with deionized water 2 Prepare anode assembly Insert the anode assembly cassette carrier into the tank
79. against a solution volume at least 40 times the sample volume for 2 h to overnight Dialyze the sample against a solution that has the same concentrations of chaotropes urea and thiourea and DTT as the sample Other more expensive solution components such as CHAPS and carrier ampholytes do not need to be included in the dialysis solution These components may be added to their required concentrations following dialysis Samples for 2 D electrophoresis should be prepared in a solution that will be compatible with first dimension IEF including urea CHAPS and DTT See section 1 6 1 for details Vivaspin Vivaspin concentrators can also be used for desalting samples 1 5 2 Removing undesirable nucleic acids from samples using Nuclease Mix Removal of nucleic acids is often required to avoid contamination and subsequent artifacts on 2 D gels Nuclease Mix offers an effective cocktail of bovine pancreatic DNase and RNase enzymes together with the necessary cofactors for optimal nuclease activity Nuclease Mix can be used together with Protease Inhibitor Mix since the latter does not contain EDTA an inhibitor of nuclease activity Protocol Nuclease Mix Components supplied Nuclease Mix 100x solution 0 5 ml Each Nuclease Mix contains 4 ug of DNase bovine pancreas and 1 ug of RNase bovine pancreas per ul solution Required but not provided Vortex mixer 1 Vortex briefly before taking an aliquot as Nuclease Mix is supplied as a
80. an Up Kit can be used for this purpose see section 1 4 1 6 3 3 Sample labeling with minimal dyes for Ettan DIGE system applications With CyDye DIGE Fluor minimal dyes it is important that primary amines e g ampholytes and thiols e g DTT are excluded from the sample until after labeling with the dyes has been completed For best results the sample concentration should be 1 10 mg ml 5 mg ml is optimal For efficient labeling the pH of the labeling reaction should be between 8 0 and 9 0 pH 8 5 is optimal A Preparation of CyDye DIGE Fluor minimal dyes for protein labeling The dimethylformamide DMF used to reconstitute the fluors should be high quality anhydrous lt 0 005 H O gt 99 8 pure It must not become contaminated with water which will start to degrade the DMF to amine compounds The DMF stock solution should be replaced at least every 3 months cP Use of molecular sieves will help keep DMF in an anhydrous condition Condensation should be prevented from forming within the fluor vials Once removed from the freezer the fluor tubes should be left for 5 min to equilibrate to room temperature prior to opening Although CyDye DIGE Fluor minimal dyes and labeled proteins are very photostable the fluors and labeled proteins should be kept covered or in the dark 1 Reconstitute CyDye DIGE Fluor minimal dyes Once the fluors have equilibrated to room temperature dispense the specified volume of DMF into the fluor vial t
81. and an external recirculating water bath such as MultiTemp III Thermostatic Circulator if temperature control is desired 3 4 1 Preparing caster and gel sandwich for miniVE SE 260 and SE 600 Ruby electrophoresis systems Protocol 1 Select gel thickness for the system Either 1 0 or 1 5 mm thick spacers can be used for all the smaller vertical formats Thinner gels stain and destain more quickly and generally give less background staining Thicker gels have a higher protein capacity Thicker gels are also less fragile and easier to handle 2 Assemble unit Mount the clamps spacers and glass plates to a sandwich Put the sandwich into the caster using the cams See instructions accompanying unit for full details 3 4 2 Preparing lab cast gels for miniVE SE 260 and SE 600 Ruby electrophoresis systems W Some of the chemicals used in the procedures acrylamide N N methylenebisacrylamide ammonium persulfate TEMED thiourea DTT and iodoacetamide are very hazardous Acrylamide monomer for example is a neurotoxin and suspected carcinogen You should have a manufacturer s safety data sheet MSDS detailing the properties and precautions for all chemicals in your laboratory These safety data sheets should be reviewed prior to starting the procedures described in this handbook General handling procedures for hazardous chemicals include using double latex gloves for all protocols Hazardous materials should be weighed in a fume
82. and chilling two to four times 1 1 3 Processing small tissue or cell samples using Sample Grinding Kit Sample Grinding Kit is designed to disrupt cell or tissue samples It utilizes an abrasive grinding resin and grinding pestle to rupture cells for protein extraction Intracellular organelles are also disrupted resulting in the liberation and extraction of all proteins soluble in the extraction solution Samples of 100 mg or less can be processed in as little as 10 min The kit contains fifty 1 5 ml microcentrifuge tubes each containing a small quantity of abrasive grinding resin suspended in water The tube is centrifuged to pellet the resin and the water is removed The methodology is outlined in Figure 8 sample Pa grinding pestle Fig 8 Schematic of the method used in the Sample Grinding Kit a Pellet grinding resin in microcentrifuge tube b Add sample and extraction solution Disrupt sample by grinding with pestle c Centrifuge to separate cellular debris and resin d Collect supernatant 80 6429 60 AD 21 The extraction solution of choice is added to the tube along with the sample to be ground A disposable pestle is supplied to grind the sample Immediately after grinding cellular debris and grinding resin are removed by 5 10 min of centrifugation If desired following extraction the sample solution may be treated to remove interfering substances using 2 D Clean Up Kit see section 1 4 1 Proto
83. andling Workstation comprises a stand alone controlled atmosphere cabinet containing a spot picker spotter digester incubator dryer microplate hotel that also stores gel trays and MS targets and robot for transferring samples between the modules A computer with proprietary software controls the whole process As an option the processing in Ettan Spot Handling Workstation can be integrated into Scierra Laboratory Workflow System LWS a communication platform for the entire 2D MS workflow This software compiles and handles information from receipt of samples through gel electrophoresis and processing in the workstation to information analysis and reporting Communication and information transfer from Ettan Spot Handling Workstation to Scierra LWS is completely automated 5 4 1 Picking protein spots Ettan Spot Picker is a robotic system that automatically picks selected protein spots from stained or destained gels using a pick list created from the image analysis software and transfers them into microplates DALT precast gels or lab cast gels are stained with Coomassie silver or fluorescent dyes and two visible reference markers are attached to each gel The gels are scanned using ImageScanner or Typhoon and analyzed using ImageMaster 2D Platinum or DeCyder 2 D Differential Analysis Software The positions of selected protein spots are exported as a pick list to Ettan Spot Picker The gels are placed into the instrument under liquid and th
84. applicable when using basic pH intervals pH 6 9 pH 6 11 and pH 7 11 NL Paper bridge loading can also be performed in the Manifold Using 18 or 24 cm Immobiline DryStrip gels up to 450 ul can be applied using the paper bridge method Details of appropriate sample loads for analytical and preparative loading and cup loading using the Manifold are given in Table 16 see section 2 5 Figure 24 gives general guidelines on selecting the appropriate mode of sample application Analytical Preparative pH gradient Strip Holder Manifold Manifold 3 5 4 5 3 0 5 6 NL 3 0 7 0 NL Cup loading 3 0 10 0 3 0 10 0 NL 3 0 11 0 NL 6 2 7 5 6 0 9 0 Cup 6 0 11 0 loading 7 0 11 0 NL Fig 24 Guidelines for selecting the appropriate mode of sample application in the Ettan IPGphor 3 Isoelectric Focusing System Refer to section 2 7 for more details on sample application 52 80 6429 60 AD 2 5 Recommended sample loads Recommended sample loads for silver for analytical analysis and Coomassie for preparative analysis staining are shown in Table 16 2 D Quant Kit see section 1 7 1 can be used to determine the protein concentration prior to first dimension IEF Table 16 Suitable sample loads for silver and Coomassie staining using cup loading and rehydration loading Immobiline DryStrip gel Suitable sample load yg of protein CyDye length cm pH
85. aration of other rehydration SOlUtIONS ceccccccccsccsccsssssssssssssssssunsssssssssececeeeeessesssnsssssass 57 2 7 Immobiline DryStrip Gel rehydration USING ACCESSOr eS wo eccsssssssssssceccsescesssssssssnustunssessssseseeeeeseees 57 2 8 Isoelectric focusing guidelines Ettan IPGphor 3 SYSTEM weecccccsssssssscccscecesssssssssssununsssseseseeceeseees 63 2 8 1 Protocol examples Ettan IPGphor 3 Isoelectric FOCUSING System 63 2 8 2 RUNNING AN Ettan IPGphor 3 ProtOCol ceeeeesssscssssescccccccccesssssssssssnssnssesesseseeeeeeeeeesesssssssssees 63 2 8 3 Preservation of focused IMMobiline DryStrip gels cccessssssssssssssssssssssseeececeeceeesesssssssnsees 67 2 9 TOUDIESMO OTN Gsis nunki A Aa ania i 68 3 Second dimension SDS PAGE using vertical electrophoresis SySteEMS scsssssssssesssececeeeeeees 71 BIOS SUM OW a ceerscdsac crac ayeata E A A E E eat Gt EE OO 71 3 1 Equilibrating IMMobiline DryStrip Gels eccecccccsssssssssssssssssssnsssessssccsssseeeceesesesssssssssssssssussnssseccecsesseeeeesees 71 3 1 1 Equilibration solution components ral 3 1 2 Equilibrating Immobiline DryStrip Gels cceccccccccccccsssssssssssssssunsssesseseeseeeeeeesessessssssnssnneneees 72 3 2 Background tO S DS PAGE vaicssesscccts2esccessastadsaecneacdaastecttdieanatttesadnanads RA 72 3 3 Electrophoresis using Ettan DALT Large Vertical electrophoresis Systems wn cscscsssscscccceceee 73 3 3 1 Preparing Ettan DALT system for electrophoresis using precast gels
86. ation of proteins through protease action greatly complicates the analysis of 2 D electrophoresis results so measures should be taken to avoid this problem If possible inhibit proteases by disrupting the sample directly into strong denaturants such as 8 M urea 10 TCA or 2 SDS 34 38 Proteases are less active at lower temperatures so sample preparation should be carried out at as low a temperature as possible In addition proteolysis can often be inhibited by preparing the sample in the presence of Tris sodium carbonate or basic carrier ampholyte mixtures These approaches alone often provide sufficient protection against proteolysis However some proteases may retain activity even under these conditions In these cases protease inhibitors may be used Individual protease inhibitors are only active against specific classes of proteases so it is usually advisable to use a combination of protease inhibitors Broad range protease inhibitor cocktails are available from a number of commercial sources GE Healthcare offers Protease Inhibitor Mix see section 1 2 1 for more details and a description of the protocol Table 7 lists common protease inhibitors and the proteases they inhibit For a more comprehensive discussion of protease inhibition see references 15 31 and 39 43 Table 7 Protease inhibitors Protease inhibitor Effective against Limitations PMSF Phenylmethylsulfonyl fluoride Most commonly used inhibitor
87. ations Table 14 shows the inter relationship between these parameters For the highest possible resolution use 24 cm strip lengths 44 80 6429 60 AD Table 14 Typical operating parameters for Immobiline DryStrip pH 4 7 gels with E coli extract and analytical load The number of detectable spots is increased by roughly the same factor as the increase in separation length The same relationship is true for other pH intervals as well Parameters 7 cm 11 cm 13 cm 18 cm 24 cm Time first dimension h 2 4 4 6 10 Time second dimension h 15 25 3 5 5 5 5 Sample load ug proteins analytical gels 10 25 30 55 90 2 2 2 Choosing the pH gradient Immobiline DryStrip gels allow effective IEF over a wide pH range from very acidic proteins at pH 3 to extremely basic proteins at pH 11 These varied pH intervals allow fine tuning of each separation strategy to increase first dimension loading and resolve a greater number of spots from crowded areas Both aspects will improve later protein identification and characterization To overview total protein distribution use pH 3 11 NL strips NL refers to nonlinear The broad range pH 3 11 NL Immobiline DryStrip gel works with most protein mixtures from prokaryotic and eukaryotic cells Results obtained can be used as a basis for developing a more specific separation strategy using medium range pH gradients For increased resolution between pH 5 and pH 7 use a nonlinear gradient pH 3 10 stri
88. bate his effect Silver or Coomassie blue staining Staining of carrier ampholytes Properly wash glass plates Scavenge any excess or residual thiol reducing agent with iodoacetamide before loading the Immobiline DryStrip gel onto the second dimension gel Use IPG Buffer as carrier ampholyte mixture Reduce concentration if necessary Prolong fixing time Background smear toward top of gel Silver staining Nucleic acids in sample Add DNase and RNase to hydrolyze nucleic acids Note The proteins DNase and RNase may appear on the 2 D map High background in top region of gel Protein contaminant in SDS electrophoresis buffer or dirty electrophoresis unit Make fresh SDS electrophoresis buffer Clean electrophoresis unit 128 80 6429 60 AD Appendix Solutions Some of the chemicals used in the procedures acrylamide N N methylenebisacrylamide ammonium w persulfate TEMED DTT iodoacetamide and DeStreak Reagent are very hazardous Acrylamide monomer for example is a neurotoxin and suspected carcinogen Read the manufacturer s safety data sheet MSDS detailing the properties and precautions for all chemicals in your laboratory These safety data sheets should be reviewed prior to starting the procedures described in this handbook General handling procedures for hazard ous chemicals include using double latex gloves for all protocols Hazardous materials should be weighed in a fume h
89. bel cysteine residues Two dyes available CyDye DIGE Fluor saturation dyes are reconstituted at 2 mM analytical gels or 20 mM preparative gels Once reconstituted the dyes are stable or up to 2 months at 15 C to 30 C Once reconstituted dyes do not need to be diluted further NHS ester dyes Label lysine residues Three dyes available Once reconstituted the concentrated stock 1 mM of CyDye DIGE Fluor minimal dyes is stable for up to 2 months at 15 C to 30 C The working concentration of the dyes is 0 4 mM and is stable for 1 week Reducing step Protein labeling Proteins must be reduced using TCEP prior to labeling Labeling reaction performed at 37 C Labeling reaction quenched using 2x sample buffer Labeling is optimized by titrating TCEP and dye Cy3 and Cy5 then analyzing ona 1 D gel Labeled proteins are stable for 1 month at 70 C No reduction step required Labeling reaction performed at 4 C Labeling reaction quenched with 10 mM lysine Labeling is optimized by comparing labeled samples on a 1 D gel Labeled proteins have stability equivalent to unlabeled protein at 70 C Protein separation and analysis No iodoacetamide equilibration step prior to 2 D electrophoresis A Cy3 labeled sample is used to prepare a preparative gel for spot picking No staining is required lodoacetamide equilibration step required An unlabeled sample is used to prepare a
90. ber stopper a piece of rigid tubing and a piece of flexible tubing Refer to the Ettan DALTsix user manual for more information 80 6429 60 AD 73 DALT gel casting cassettes DALT gel casting cassettes fit either Ettan DALTsix or Ettan DALTtwelve electrophoresis units In the standard hinged cassette one tall and one short glass plate are hinged together with vinyl spacers glued in place The simplified design of the cassette allows for easy assembly and no clamps are required to seal the cassette Standard glass plate sets and low fluorescent glass plate sets are available for use with Ettan DALT systems Vinyl spacers are glued into place as with the standard cassette The plates are not hinged which means that a single glass plate is able to fit into the spot picker when the gel is chemically bound to the plate using Bind Silane see appendix V DALT Gel 12 5 and DALT Precast Gel Cassette DALT Gel 12 5 is a precast polyacrylamide gel 25 5 x 19 6 cm 1 mm thick for the second dimension of 2 D electro phoresis The gel is provided already cast onto a plastic support film The gel is a homogeneous 12 5 polyacrylamide gel It is intended to be used in Ettan DALTsix or Ettan DALTtwelve system together with the DALT Buffer Kit The gel is formulated for long shelflife and when used with the buffer kit generates a discontinuous buffer system yielding rapid runs with sharp reproducible results The gels are inserted into a specially designed reus
91. bubbles are trapped between the Immobiline DryStrip gel and the slab gel surface or between the gel backing and the glass plate 3 Optional Apply molecular weight marker proteins Best results are obtained when the molecular weight marker protein solution is mixed with an equal volume of a hot 1 agarose solution prior to application to the IEF sample application piece The resultant 0 5 agarose will gel and prevent the marker proteins from diffusing laterally prior to the application of electric current Other alternatives are to apply the markers to a paper IEF sample application piece in a volume of 15 20 ul For less volume cut the sample application piece proportionally Place the IEF application piece on a glass plate and pipette the marker solution onto it then pick up the application piece with forceps and apply to the top surface of the gel next to one end of the Immobiline DryStrip gel The markers should contain 200 1000 ng of each component for Coomassie staining and approximately 10 50 ng of each component for silver staining 4 Seal the Immobiline DryStrip gel in place The agarose sealing solution prevents the Immobiline DryStrip gel from moving or floating in the electrophoresis buffer For precast DALT gels the agarose blocks the narrow gapls between the gel edgels and the lateral spacer s to prevent leakage of the upper buffer Prepare agarose sealing solution for DALT precast gels using the agarose sealing solution from the
92. capacity two dimensional gel electrophoresis Electrophoresis 21 3649 3656 2000 Bjellqvist B et al A nonlinear wide range immobilized pH gradient for two dimensional electrophoresis and its definition in a relevant pH scale Electrophoresis 14 1357 1365 1993 Gorg A et al 2 D electrophoresis with immobilized pH gradients using IPGphor isoelectric focusing system Life Science News 1 4 6 1998 Ibel K et al Protein decorated micelle structure of sodium dodecyl sulfate protein complexes as determined by neutron scattering Eur J Biochem 190 311 318 1990 Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227 680 685 1970 Schdgger H and von Jagow G Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa Anal Biochem 166 368 379 1987 Gorg A et al Elimination of point streaking on silver stained two dimensional gels by addition of iodoacetamide to the equilibration buffer Electrophoresis 8 122 124 1987 Shevchenko A et al Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels Anal Chem 68 850 858 1996 Neuhoff V et al Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels a systematic analysis Electrophoresis 6 427 448 1985 Neuhoff V et al Improved staining of proteins in polyac
93. ch as CHAPS SDS or Triton X 100 Assays that depend on the reduction of cupric ions cannot be used in the presence of reductants such as DTT or in the presence of reagents that form complexes with cupric ions such as thiourea or EDTA Samples prepared for IEF and SDS gel electrophoresis are often difficult to quantitate due to the presence of detergent and reductant Samples for 2 D electrophoresis are particularly difficult to quantitate due to the possible presence of interfering carrier ampholyte and thiourea in addition to the detergents and reductants typically used in sample preparation 2 D Quant Kit designed for the accurate determination of protein concentration in samples to be analyzed by high resolution electrophoresis circumvents these limitations and can be used to accurately quantitate protein samples prepared for 2 D electrophoresis The procedure uses a combination of a unique precipitant and co precipitant to quantitatively precipitate sample protein while leaving interfering contaminants in solution The protein is pelleted by centrifugation and resuspended in an alkaline solution of cupric ions The cupric ions bind to the polypeptide backbones of any protein present A colorimetric agent that reacts with unbound cupric ions is then added The color density is inversely related to the concentration of protein in the sample and the protein concentration can be accurately estimated by comparison to a standard curve Since the assay d
94. cision shock on the protein metabolism of Vicia faba L meristematic root cells Planta 155 478 485 1982 Wolpert T J and Dunkle L D Alternations in gene expression in sorghum induced by the host specific toxin from Periconia circinata Proc Natl Acad Sci USA 80 6576 6580 1983 Blomberg A et al Interlaboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient two dimensional gel electrophoresis Electrophoresis 16 1935 1945 1995 Damerval C et al Technical improvements in two dimensional electrophoresis increase the level of genetic variation detected in wheat seedling proteins Electrophoresis 7 52 54 1986 Wu F S and Wang M Y Extraction of proteins for sodium dodecyl sulfate polyacrylamide gel electrophoresis from protease rich plant tissues Anal Biochem 139 100 103 1984 Harrison P A and Black C C Two dimensional electrophoretic mapping of proteins of bundle sheath and mesophyll cells of the C4 grass Digitaria sanguinalis Plant Physiol 70 1359 1366 1982 Granzier H L M and Wang K Gel electrophoresis of giant proteins solubilization and silver staining of titin and nebulin from single muscle fiber segments Electrophoresis 14 56 64 1993 Colas des Francs C et al Analysis of leaf proteins by two dimensional gel electrophoresis Plant Physiol 78 178 182 1985 Barret A J and Salversen G Proteinase Inhibitors Elsevier Press Amsterdam 1986 No
95. col Sample Grinding Kit Components supplied Microcentrifuge grinding tubes containing grinding resin suspended in water disposable pestles for sample grinding Required but not provided Microcentrifuge capable of at least 12000 x g vortex mixer extraction solution Preliminary notes Samples can be extracted into 8 M urea and 4 CHAPS or into 7 M urea 2 M thiourea and 4 CHAPS see solutions A and B in appendix Alternative nonionic detergents or protease inhibitors can be added during extraction Carrier ampholytes Pharmalyte reagents Ampholines or IPG Buffers can be added at concentrations up to 2 for standard protocols but should not be added during protein extraction for labeling in 2 D DIGE 1 Briefly centrifuge the grinding resin at maximum speed in the 1 5 ml microcentrifuge tubes provided in the kit Fig 8A Remove supernatant with micropipette 2 Add sample up to 100 mg and extraction solution of choice 200 300 ul see appendix solutions A and B Tissue can be cut up with a scalpel or frozen with liquid nitrogen and broken with mortar and pestle to yield tissue fragments Cell suspensions can be centrifuged with the grinding resin and resuspended in extraction solution 3 Grind sample thoroughly up to 1 min with the disposable pestle included in the kit Fig 8B 4 Limit extraction solution to 200 300 ul during grinding to prevent liquid from splashing out of the tube Additional extraction soluti
96. cts the strips from dust and other contaminants during the rehydration period which ranges from 10 h to overnight 2 3 4 Ettan IPGphor 3 Strip Holders IPGphor Strip Holder serves as both a rehydration and focusing chamber for individual IPG strips When the sample is included in the rehydration solution it is loaded into the gel by absorption during the rehydration step Since the gel is in direct contact with electrodes built into the Strip Holder it is placed in position to run without further handling The base of the Strip Holder is made from the same thermally conductive aluminum oxide ceramic as the Manifold and has platinum electrodes at each end The transparent Strip Holder cover allows easy visual monitoring of rehydration and focusing progress 2 3 5 General cautions Ettan IPGphor 3 is a high voltage instrument that can cause fatal electrical shock if the safety features are disabled As such the safety lid must be properly latched before starting a protocol otherwise voltage will not be applied Exceeding the recommended current limit of 75 yA per IPG strip can cause the strip to burn and may damage the instrument During isoelectric focusing do not lean on the safety lid do not apply excess pressure or uneven weight to the lid and do not place any items on the lid Such pressure could cause arcing between the Strip Holder electrodes and the electrode areas damaging the instrument The Strip Holders and Manifold trays are ma
97. d See Table 17 for buffers Table 17 Immobiline DryStrips and IPG buffers Immobiline DryStrip IPG Buffer PH 3 5 4 5 3 5 6 NL 3 5 5 0 pH 3 7 NL 4 7 4 7 pH 3 10 3 10 pH 3 10 NL 3 10 NL pH 3 11 NL 3 11 NL pH 5 3 6 5 5 5 6 7 pH 6 9 6 11 6 2 7 5 6 11 pH 7 11 NL 7 11 NL Rehydration of Immobiline DryStrips 1 Pipette the appropriate volume of prepared DeStreak Rehydration Solution into the Reswell Tray or into the regular Strip Holder as indicated in Table 18 Distribute the solution evenly over the channel length 2 Carefully remove the cover foil from the Immobiline DryStrip starting from the anodic end end 3 Carefully place the Immobiline DryStrip into the tray holder channel gel side down Take care to distribute the rehydration solution evenly under the strip To help coat the entire gel gently lift and lower the strip and slide it back and forth along the surface of the solution Be careful not to trap bubbles under the Immobiline DryStrip gel 4 Close the lid to the IPGbox 5 Rehydrate for 10 20 h Sample application 1 Load the sample either in the rehydration solution or after rehydration using a sample cup or anodic paper bridge e With acidic pH intervals 3 5 4 5 and 3 5 6 NL we recommend rehydration loading or cathodic sample cup application Use up to 20 mM reducing agent per 100 ul of sample e With neutral 5 3 6 5 and 4 7 and wide 3 10 pH intervals al
98. d and very low currents typically less than 50 pA per Immobiline DryStrip gel due to the low ionic strength within Immobiline DryStrip gels During IEF the current decreases while the voltage increases as proteins and other charged components migrate to their equilibrium positions A typical IEF protocol generally proceeds through a series of voltage steps that begins at a relatively low value Voltage is gradually increased to the final desired focusing voltage which is held for several hours A low initial voltage minimizes sample aggregation and allows the parallel separation of samples with differing salt concentrations A gradual increase in voltage is particularly advisable for higher protein loads 100 ug or more per Immobiline DryStrip gel Many factors affect the amount of time required for complete focusing and each specific set of conditions e g sample and rehydration solution composition Immobiline DryStrip gel length and pH gradient requires empirical determination for optimal results An approximate time for complete focusing is given in the example protocols provided in Table 19 Factors that increase the required focusing time include residual ions which must move to the ends of the Immobiline DryStrip gels before protein focusing can occur and the presence of IPG Buffers or Pharmalyte which contributes to the ionic strength of the electrophoresis medium A higher IPG Buffer concentration increases the conductivity of the Immobil
99. d a protocol for use of this reagent DeStreak Rehydration Solution contains DeStreak Reagent as described above The Rehydration Solution also contains optimized concentrations of urea thiourea and CHAPS and is ready for use after addition of the appropriate IPG Buffer IPG Buffer or Pharmalyte carrier ampholyte mixtures improves separations particularly with high sample loads Carrier ampholyte mixtures enhance protein solubility and produce more uniform conductivity across the pH gradient without disturbing IEF or affecting the shape of the gradient IPG Buffers are carrier ampholyte mixtures specially formulated not to interfere with silver staining following 2 D electrophoresis Select an IPG Buffer with the same pH interval as the Immobiline DryStrip to be rehydrated Table 17 The advantages of increased concentration of IPG Buffer Pharmalyte are e Improved sample solubilization e Increased tolerance to salt in sample e More even conductivity in the gel EF Higher concentrations of IPG Buffer Pharmalyte will limit the voltage usable during IEF and increase the time required for the focusing step Er Silver staining may require a prolonged fixing step to wash out carrier ampholyte that may cause staining background near the ion front of the second dimension gel cr IPG Buffer or Pharmalyte can be included in the stock rehydration solution or added just prior to use The carrier ampholytes are included in the stock solution when m
100. d regardless of whether the Manifold is being used the maximum voltage is 8000 V Table 19 Guidelines for running 7 24 cm Immobiline DryStrip gels on Ettan IPGphor 3 Isoelectric Focusing Unit Running conditions Temperature 20 C current 50 pA per strip except where noted See footnotes for information specific to the different strip lengths 7 cm strips pH Voltage mode Voltage Time kVh intervals Vv h min 3 11 NL 1 Step and Hold 300 0 30 0 2 3 10 2 Gradient 1000 0 30 0 3 6 11 3 Gradient 5000 1 20 4 0 4 Step and Hold 5000 0 06 0 25 0 5 2 0 Total 2 26 2 45 5 0 6 5 3 10 NL 1 Step and Hold 300 0 30 0 2 4 7 2 Gradient 1000 0 30 0 3 3 5 6 NL 3 Gradient 5000 1 30 4 5 4 Step and Hold 5000 0 12 0 36 1 0 3 0 Total 2 42 3 06 6 0 8 0 7 11 NL 1 Step and Hold 300 0 30 0 2 2 Gradient 1000 1 00 0 7 3 Gradient 5000 1 30 45 4 Step and Hold 5000 0 20 0 55 1 6 4 6 Total 3 20 3 55 7 0 10 0 5 3 6 5 1 Step and Hold 300 1 00 0 2 6 2 7 5 2 Gradient 1000 1 00 0 7 3 Gradient 5000 2 30 75 4 Step and Hold 5000 0 45 1 30 3 6 7 6 Total 5 15 6 00 12 0 16 0 64 80 6429 60 AD Table 19 continued 11 cm strips pH Step Voltage Voltage Time kVh intervals mode Vv h min 3 11 NL 1 Step and Hold 500 1 00 0 5 3 10 2 Gradient 1000 1 00 0 8 6 11 3 Gradient 6000 2 00 7 0 4 Step and Hold 6000 0 10 0 40 0 7 3 7 Total 4 10 4 40 9 0 12 0 4 7 1 Step and Hold 500 1 00
101. d serves as an electrical insulating fluid to ensure good thermal contact between the cooling plate and the tray Connect the red and black electrode leads on the tray to the Multiphor II unit Position the Immobiline DryStrip aligner Pour approximately 10 ml of Immobiline DryStrip Cover Fluid into the Immobiline DryStrip tray Place the Immobiline DryStrip aligner 12 groove side up into the tray on top of the Immobiline DryStrip Cover Fluid The presence of air bubbles between the strip positions under the aligner will not affect the experiment For easier visualization of the grooves in the aligner avoid getting Immobiline DryStrip Cover Fluid on top of the aligner B Prepare electrode strips 1 Cut electrode strips to size Cut two IEF electrode strips to lengths of 110 mm each Soak electrode strips with distilled water Place the electrode strips on a clean flat surface such as a glass plate Soak each electrode strip with 0 5 ml distilled water Blot with filter paper to remove excess water EF Electrode strips must be damp not wet Excess water may cause streaking C IEF with rehydration loading ale Remove the rehydrated Immobiline DryStrip gel from the Immobiline DryStrip IPGbox To remove an Immobiline DryStrip gel from the Immobiline DryStrip IPGbox slide the tip of a pair of forceps along the sloped end of the channel and into the slight depression under the Immobiline DryStrip gel Grasp the end of the strip with
102. d the top surface of the second dimension gel Flatbed gel format Urea crystals on the surface of the Immobiline DryStrip gel Allow residual equilibration solution to drain from the Immobiline DryStrip gel before placing the strip on the second dimension gel Flatbed gel format Bubbles under the Immobiline DryStrip gel Ensure that the Immobiline DryStrip gel is placed firmly on the gel with no air bubbles trapped underneath Stroke the plastic backing of the Immobiline DryStrip gel gently with a pair of forceps to remove trapped bubbles continues on following page 80 6429 60 AD 127 Table 44 Troubleshooting 2 D results continued Symptom Possible cause Remedy Poor representation of higher molecular weight proteins Proteolysis of sample Prepare sample in a manner that limits proteolysis and or use protease inhibitors see section 1 2 nsufficient equilibration Prolong equilibration time Poor transfer of protein from mmobiline DryStrip gel to second dimension gel Employ a low current sample entry phase in the second dimension electrophoresis run Poor entry of sample protein during rehydration Use recommended volume of rehydration solution Table 18 Point streaking Background smear toward bottom of gel Silver staining Dirty plates used to cast gel or particulate material on the surface of he gel DTT and other thiol reducing agents exacer
103. de of ceramic and should be handled carefully Always wear gloves when handling IPG strips and the equipment that comes in contact with them This will help minimize protein contamination which can result in artifactual spots in the resulting 2 D spot patterns Clean Strip Holders and Manifold with the Strip Holder cleaning solution provided or the protective coating will be compromised Clean all other components that come in contact with the IPG strip or the sample with a detergent designed for glassware Rinse well with distilled water Use the appropriate rehydration volume for the IPG strip length refer to appropriate protocol Do not heat any solutions containing urea above 30 C as isocyanate a urea degradation product will carbamylate the proteins in the sample thus changing their isoelectric points OOO GOS SG G All chemicals should be of the highest purity electrophoresis grade or better and water should be double distilled or deionized 80 6429 60 AD 51 2 4 Selecting sample application method Sample can be applied either by including it in the rehydration solution rehydration loading or by applying it directly to the rehydrated Immobiline DryStrip gel via sample cups or a paper bridge 2 4 1 Rehydration loading Rehydration loading see section 2 7 offers such advantages as loading and separation of larger sample volumes greater than 100 ul 70 71 larger sample amounts and more dilute samples Because there i
104. decreasing by 33 the amount of ammonium persulfate and TEMED used Ensure that there is no leakage during gel casting Horizontal streaking or incompletely Sample applied at too Increase the concentration of IPG buffer focused spots anodic sample application acidic pH in sample and Immobiline DryStrip in which the problem is visible at the Add slightly more alkaline IPG buffer to anodic end of the IPG strip the sample Apply the sample at the cathode Note Repeated precipitation resolubilization cycles produce or increase horizontal streaking See section 1 6 for general guidelines for sample solubilization Horizontal streaking or incompletely Sample is poorly soluble in Increase the concentration of the solubilizing focused spots rehydration loading rehydration solution components in the rehydration solution see section 2 6 Increase concentration of IPG Buffer Underfocusing Focusing Prolong focusing time time was not long enough to achieve steady state focusing Horizontal streaking or incompletely focused Interfering substances Modify sample preparation to limit these spots all sample application methods Non protein impurities inthe contaminants see section 1 4 sample can interfere with IEF Use 2 D Clean Up Kit section 1 4 1 causing horizontal streaking The effect of ionic impurities can be reduced by modifying the IEF protocol Limit the voltage to 100 150 V for 2 h then re
105. dic end of the Immobiline DryStrip gel and the electrode 375 500 ul sample can be applied using the paper bridge pads supplied with the Manifold Solutions containing up to 5 mg of protein have been loaded on an 18 cm narrow pH range Immobiline DryStrip gel 74 A standard paper electrode pad between the paper bridge and the electrode improves sample transfer and gel results 80 6429 60 AD 57 The rehydrated Immobiline DryStrip gel is first positioned in the bottom of the Manifold channel gel side up Then the paper bridge with sample is positioned followed by a paper wick With anodic application the anode electrode is positioned as far out as possible in the electrode assembly while the cathode electrode is positioned close to the end of the Immobiline DryStrip gel to ensure good contact between the paper wick and Immobiline DryStrip gel EF The application point anodic or cathodic is an important factor for obtaining good results EF A single paper bridge can be used with the 24 cm gel strip If so desired a paper bridge can be used on both ends of all other strips at one time Protocol Using the Strip Holder for gel rehydration IPGphor fixed length Strip Holders allow IPG strips to be rehydrated and samples loaded in one step before proceeding automatically to perform the IEF separation The IPG strips are 3 mm wide and 0 5 mm thick after rehydration This protocol applies for both in gel sample rehydration and sample application
106. dient the appropriate percentage gel is selected according to the range of separation desired Table 24 Table 24 Recommended acrylamide concentrations for protein separation Acrylamide percentage in resolving gel Separation size range M x 10 Single percentage 5 36 200 8 24 200 10 14 200 12 5 14 100 15 14 60 Gradient 5 15 14 200 5 20 10 200 10 20 10 150 Larger proteins fail to move significantly into the gel 2 Select gel thickness and calculate casting solution volume DALT gel casting cassettes with either 1 0 or 1 5 mm thick spacers can be used Thinner gels stain and destain more quickly and generally give less background staining Thicker gels have a higher protein capacity Thicker gels are also less fragile and easier to handle Table 25 gives the volumes required for Ettan DALT systems Table 25 Volumes required per cast Ettan DALT systems Casting system Volume ml Ettan DALTsix 6 gels x 1 mm thick spacers 450 6 gels x 1 5 mm thick spacers 600 Ettan DALTtwelve 14 gels x 1 mm thick spacers 900 13 gels x 1 5 mm thick spacers 1200 3 Calculate the formulation of the gel solution The recipes given in Table 26 produce 900 ml of solution for a single percentage gel The recipes in Table 27 produce 450 ml each of light and heavy solution for a gradient gel These recipes can be scaled up or down depending on the volume required 4 Prepare the gel solution Make
107. ding on the size range separated by the second dimension gel Pepstatin does not inhibit any proteases that are active at pH 9 TLCK TPCK Tosyl lysine chloromethyl ketone tosyl phenylalanine chloromethyl ketone Use at 0 1 0 5 mM These compounds irreversibly inhibit many serine and cysteine proteases Benzamidine Use at 1 3 mM Benzamidine inhibits serine proteases 80 6429 60 AD 23 1 2 1 Protease inhibition using Protease Inhibitor Mix Protease Inhibitor Mix from GE Healthcare contains an optimized concentration of competitive and noncompetitive protease inhibitors that effectively inhibit serine cysteine metalloproteases and calpain proteases The kit is suitable for the protection of proteins during purification from animal tissues plant tissues yeast and bacteria Protocol Protease Inhibitor Mix Reagents supplied Protease Inhibitor Mix 100x solution 1 ml Required but not provided Microcentrifuge vortex mixer extraction solution Preliminary notes Protease Inhibitor Mix is provided free of EDTA as some proteins require divalent cations such as Ca Mg or Mn for their biological activity In such circumstances the presence of EDTA may be detrimental to sample protein activity Samples can be extracted into 8 M urea and 4 CHAPS or into 7 M urea 2 M thiourea and 4 CHAPS see solutions A and B in appendix Alternative nonionic detergents or protease inhibitors can be added during
108. e Ultracentrifugation will remove high molecular weight polysaccharides Employing the same methods used for preventing protein nucleic acid interactions may also be helpful solubilize sample in SDS or at high pH Lipids Many proteins particularly membrane proteins are complexed with lipids This reduces their solubility and can affect both the pl and the molecular weight Lipids form complexes with detergents reducing the effectiveness of the detergent as a protein solubilizing agent When extracts of lipid rich tissues are centrifuged there is often a lipid layer that can be difficult to remove Strongly denaturing conditions and detergents minimize protein lipid interactions Excess detergent may be necessary Precipitation with acetone removes some lipid Phenolic compounds Phenolic compounds are present in many plant tissues and can modify proteins through an enzyme catalyzed oxidative reaction 43 49 Prevent phenolic oxidation by employing reductants during tissue extraction e g DTT 2 mercaptoethanol sulfite ascorbate Rapidly separate proteins from phenolic compounds by precipitation techniques Inactivate polyphenol oxidase with inhibitors such as diethyldithiocarbamic acid or thiourea Remove phenolic compounds by adsorption to polyvinylpyrrolidone PVP or polyvinylpoly pyrrolidone PVPP continues on following page 80 6429 60 AD 31 Table 11 Contaminants that affect 2 D result
109. e interfering substances that affect 2 D results Refer to section 1 4 1 for a discussion of 2 D Clean Up Kit which selectively precipitates protein for 2 D analysis v Salt contamination is the most frequent cause of insufficient focusing of protein spots Table 11 Contaminants that affect 2 D results Contaminant Reason for removal Removal techniques Salts residual buffers and other charged small molecules that carry over from sample preparation Salts disturb the electrophoresis process and must be removed or maintained at as low a concentration as possible Salts in the IPG strip result in high strip conductivity Focusing of the proteins will not occur until the ions have moved to the ends of the strips prolonging the time required for IEF Water movement can also occur causing one end of the strip to dry out and the other end to swell Salt in the PG strip can result in large regions at either end of the IPG strip where proteins do not focus seen as horizontal streaking or empty regions in the final result the sample is rehydrated into the IPG strip the salt concentration in the rehydration solution should be lower han 10 mM the sample is applied in sample cups salt concentrations of up to 50 mM in the sample may be tolerated however proteins may precipitate at the sample application point as they abruptly move into a lower salt environment Desalting can be performed by e
110. e 8 A First and second dimension electrophoresis with optimized Systems we cscsscsccccsceseee 9 B First and second dimension electrophoresis with a flatbed SYStOM ccccccccccccccscssssssssseenee 11 EguipmMEnt CNOICE Sieninis ipae nn a a Aan nia 12 Selecting an IEF SySte M a csscccssscsssosescenssssisnssssisostesseesdassecssscdannsesisnssnsinosossisonescsnccessdcatspssoncbosivessvaiueisnsiesdoneiaesese 12 Selecting a second diMenSion SYSTEM woaccccccsscsssssssccssssesssssssssnsntnssssessssesseececeesessssssstsnssumussssssssesseeeeeeees 13 Vertical SYSTEMS iinan anae nn i arai ian R an NEN Eai Multiphor II Electrophoresis System GOO laborat rny Practici ssimereneneinani giii E NA EEREN N A ENE 1 Sample preparation asicsccccstsiseciccsisscscccecasscasovessiecienses sondsaceascessnss esasicasssseessusassenendssoniencaceseesdueaicesbbeivenasts 17 1 0 General Strate Gy essssssssseessssssscssssesccccccsessssssnssssnsnmunesssesesseseeseeseesessssssnsnsnneese 17 1 0 1 Cell disruption protection from proteolysis fractionation ee esscsscscccccecccessesessseneeee 17 1 0 2 Precipitation and removal of interfering SUDStANCES sessssssssssseessssesessecceeceececessessssssnsees 17 1 0 3 Additional aspects of sample preparation wa eeecccccccscssccccccesssssssssssnsseesesseseseceeseeessesssssssanes 18 1 0 4 General sample preparation QUICELINES eeesssesessssssseceecececseceesssssssssssessessesseeeeeeeeee 19 AT Methods OPE GISHUPE OM see i
111. e camera detects the reference markers Control software converts spot pixel co ordinates into picking co ordinates and the Ettan Spot Picker selects and transfers gel plugs into 96 well microplates 5 4 2 Digesting proteins and spotting onto MALDI ToF MS slides The gel plugs are digested in Ettan Digester the supernatant peptides are mixed with matrix assisted laser desorption ionization time of flight mass spectrometry MALDI ToF MS matrix material and spotted onto MALDI ToF MS slides using Ettan Spotter 5 4 3 MALDI ToF mass spectrometry Time of flight mass spectrometry is a technique for analyzing molecular weights based on the motion of ionized samples in an electrical field In Ettan MALDI ToF Pro mass spectrometer a matrix bound sample is bombarded with a pulsed laser beam to generate ions for subsequent detection Ettan MALDI ToF Pro provides fast and precise identification of proteins in high throughput peptide mass fingerprinting PMF 108 80 6429 60 AD The novel quadratic field reflectron Z reflectron technology in Ettan MALDI ToF Pro offers single run post source decay PSD data acquisition in approximately 1 min Automated database searching of PSD data allows rapid and precise protein identification from single tryptic peptides In cases where PMF cannot provide unambiguous protein identification reliable information can be obtained by using the instrument in conjunction with CAF MALDI Sequencing Kit Chemically assisted
112. e difficult to resuspend 14 Resuspend each pellet in an appropriate volume of rehydration or IEF sample loading solution for first dimension IEF 1 See next section for examples of rehydration solutions and volumes appropriate to different applications Vortex the tubes for at least 30 s Incubate at room temperature Vortex or aspirate and dispense using a pipette to fully dissolve EF If the pellet is large or too dry it may be difficult to resuspend fully Sonication or treatment with the Sample Grinding Kit see section 1 1 3 can speed resuspension Sa Centrifuge the tubes at maximum speed at least 12 000 x g for 5 min to remove any insoluble material and to reduce any foam The supernatant may be loaded directly onto first dimension IEF or transferred to another tube and stored at 80 C for later analysis B For larger samples of more than 100 ug of protein All steps should be carried out on ice unless otherwise specified il 1 1 1 28 Transfer the protein samples into tubes that can be centrifuged at 8000 x g Each tube must have a capacity at least 12 fold greater than the volume of the sample Use only polypropylene polyallomer or glass tubes The wash buffer used later in the procedure is not compatible with many plastics This limits the choice of centrifuge tube materials For each volume of sample add three volumes of precipitant Mix well by vortexing or inversion Incubate on ice 4 5 C for
113. e electrode connectors and carefully lower the electrode holder onto the buffer strips Fig 56 Fig 55 Positioning application pieces Fig 56 Positioning electrodes 4 2 3 Electrophoresis conditions Place the safety lid on the Multiphor II unit Connect the power supply Recommended electrical settings and running times are listed in Table 39 Table 39 Electrophoresis conditions for ExcelGel gels Step Voltage V Current mA Power W Duration h min ExcelGel SDS Homogeneous 12 5 1 120 20 30 0 40 Open the lid and carefully remove the electrodes 2 600 50 30 1 10 ExcelGel Gradient XL 12 14 1 200 20 20 0 40 Open the lid and carefully remove the electrodes 2 800 40 40 2 40t Remove the Immobiline DryStrip gel and the application pieces Then move the cathodic buffer strip forward to cover the area of the removed Immobiline DryStrip gel Adjust the position of the cathodic electrode Stop electrophoresis 5 min after the bromophenol blue front has reached the anodic buffer strip Remove and discard the buffer strips 80 6429 60 AD 103 4 2 4 Troubleshooting L Table 40 lists possible problems that could be encountered during second dimension SDS PAGE using the Multiphor II Electrophoresis System and how to solve them Table 40 Troubleshooting second dimension SDS PAGE Multiphor II Electrophoresis System Symptom Possible cause Remedy No current at start of run Dye front curves up smiles at
114. e fully contacts the dialysis solution and that no large air bubbles are trapped beneath the dialysis membrane Remove any air bubbles by tilting the tube or squirting dialysis solution onto the membrane Ee See section below on dialysis solutions 6 Dialyze while stirring Fig 11C During dialysis invert dialysis tube to thoroughly mix contents Note Optimal dialysis time depends on several factors including the nature and volume of the sample the molecular weight cut off of the dialysis membrane and the temperature Normally dialysis for 2 h to overnight is sufficient to reduce ionic contaminants to a level that does not interfere with IEF separation Dialysis may be carried out at 4 8 C to minimize sample degradation or modification but this will slow down the dialysis Dialysis can be conducted at room temperature if degradation or modification is not a concern 7 After dialysis centrifuge dialysis tube for 6 s at 500 1000 x g to collect sample Fig 11D 8 Remove dialysis cap and replace with normal cap for storage The protein concentration of the sample is best determined using the 2 D Quant Kit The kit allows accurate quantitation of protein in the presence of detergents reductants and chaotropes that are incompatible with other assays See section 1 7 1 for a protocol describing the use of 2 D Quant Kit 80 6429 60 AD 33 Dialysis solution A substantial reduction in interfering ions can be achieved by dialyzing 2 D samples
115. e loaded directly onto first dimension IEF or transferred to another tube and stored at 80 C for later analysis 1 4 2 Resuspension of pellet 2 D Clean Up Kit produces a protein pellet When using cup loading resuspend the pellet in sample preparation solution see appendix When using rehydration loading resuspend the pellet in rehydration solution see options 1 and 2 below which is applied directly to the Immobiline DryStrip gel 1 Rehydration solution containing 8 M urea Use solution C in appendix This all purpose solution gives clean sharp 2 D separations 2 Rehydration solution containing 7 M urea and 2 M thiourea Use solution D in appendix This is a more strongly solubilizing solution that results in more spots in the final 2 D pattern Any other components added to the rehydration solution must either be uncharged or present at a concentration of less than 5 mM The addition of salts acids bases and buffers is not recommended 3 DeStreak Reagent Use for basic strips See section 2 6 2 for details on the reagent Sample resuspension volumes The volume of rehydration solution used to resuspend the sample depends on the sample loading method and the length of the Immobiline DryStrip gel used for the first dimension separation If using Ettan IPGphor 3 and the sample is to be loaded onto the Immobiline DryStrip gel using a sample cup the sample volume should not exceed 150 ul If the sample is to be loaded onto the
116. e manual adjustment of the strip may be necessary Fig 30 Note If cathodic cup loading is going to be used the strips should be placed such that the anodic end of the strips is 3 4 cm beyond the etched placement mark If performing cup loading place a strip of cups in the appropriate position Fig 31 for example 1 cm from the end of the gel portion of the IPG strip Do NOT place the cup with the feet over a center protrusion Push the cups into the channels with gloved fingers starting at one end and working toward the other Align the insertion tool over the cups and push down to ensure that the feet of the cups are properly seated at the bottom of the channel wiggle the tool gently while pushing down in order to ensure that the cups are seated as far down as they will go Take care not to move the cups while removing the insertion tool Ensure that the cups are filled with cover fluid EF If desired test for leakage by adding some colored sample buffer without sample If no leaks are detected pipette the colored liquid back out again ire Cups must not straddle the centering protrusions on the bottom of the channels Count out the appropriate number of precut paper wicks Two wicks per strip are required Separate the wicks from each other Add 150 ul of distilled water to each wick Place the wicks on the IPG strips such that one end of the wick overlaps the end of the gel on the IPG strip Fig 32 For gradients with pH above
117. e red or black circle Ensure that the bridging cable under the cooling plate is properly installed Immobiline DryStrip gels are improperly rehydrated Ensure that the Immobiline DryStrip gels are rehydrated along its entire length The high voltage lead from he electrophoresis unit is supply correctly Ensure that the plugs on the high voltage leads fit securely into the output jacks on the power supply Use the appropriate not plugged into the power adapter if necessary Sample dye does not move out of the sample cup It is normal dye leaves for several hours o elapse before the sample he sample cups down so ha rest agains This blocks The sample cups were pressed rd against the gel that hey pushed through the gel to the plastic backing the current and physically prevents the protein rom entering the Immobiline DryStrip ge S Replace Immobiline DryStrip gel and re apply sample cup The ionic st rength of the sample is higher than the gel As a result he field strength in the sample zone is inadequate to move the protein out of the sample zone at an appreciable rate Dilute the sample as much as possible or just prior to loading dialyze the sample to remove salts Sparking or burning of Immobiline DryStrip gels Conductivity of the sample Immobiline DryStrip gel is too high Ensure the sample is adequately desalted Alternatively be
118. e sample representation or a clear reproducible pattern Additional sample preparation steps can improve the quality of the final result but each additional step can result in the selective loss of protein species The trade off between improved sample quality and complete protein representation must therefore be carefully considered In order to characterize specific proteins in a complex protein mixture the proteins of interest must be completely soluble under electrophoresis conditions Different treatments and conditions are required to solubilize different types of protein samples some proteins are naturally found in complexes with membranes nucleic acids or other proteins some proteins form various nonspecific aggregates and some proteins precipitate when removed from their normal environment The effectiveness of solubilization depends on the choice of cell disruption method protein concentration and dissolution method choice of detergents and composition of the sample solution If any of these steps are not optimized for a particular sample separations may be incomplete or distorted and information may be lost 1 0 1 Cell disruption protection from proteolysis fractionation To fully analyze all intracellular proteins the cells must be effectively disrupted Choice of disruption method depends on whether the sample is derived from cell suspensions solid tissue or other biological material and whether the analysis is targeting all pr
119. e the technology for commercial purposes is subject to a separate license agreement with GE Healthcare Bio Sciences AB Please contact the Product Director Mass Spectrometry and Sample Handling GE Healthcare Bio Sciences AB Bj rkgatan 30 SE 75184 Uppsala Sweden for details about how to obtain such a license This version of ImageMaster has been developed by the Swiss Institute of Bioinformatics in collaboration with GeneBio and GE Healthcare All third party trademarks are the property of their respective owners 2010 General Electric Company all rights reserved All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 80 6429 60 AD 06 2010
120. ed off Begin IEF EF As isoelectric focusing proceeds the bromophenol blue tracking dye migrates toward the anode Note that the dye front leaves the Immobiline DryStrip gel well before focusing is complete so clearing of the dye is no indication that the sample is focused If the dye does not migrate no current is flowing If this occurs check the contact between the electrodes and the electrode strips Also check that the electrode leads and bridging cable are correctly connected and check that the electrodes are posi contacts the side rail The protocols below are suitable for running 7 24 cm Immobiline DryStrip ge System connected to EPS 3501 XL Power Supply ioned properly so that the marked side s on the Multiphor II Electrophoresis The focusing times given in Table 37 are guidelines only They may vary with the nature of the sample and how the sample is applied If using crude samples with high protein and sa the run time in total kilovolt hours should be increased by 10 t content or using paper bridge loading Table 37 Guidelines for running Immobiline DryStrip gels on Multiphor II Electrophoresis System Running conditions Temperature 20 C current 2 mA total power 5 W total Program EPS 3501 XL Power Supply in gradient mode and with current check option turned off 7 cm strips pH intervals Step Voltage V Time h kVh 3 11 NL 1 200 0 01 3 10 2 3500 1 30 2 8 6 11 3 3500
121. ed using lint free cloths such as Crew Wipes e During the protein staining step a volume of working stain solution equivalent to at least a 10 fold excess of the gel volume should be used During all other steps a volume equivalent to 20 fold excess of the gel volume should be used Table A e Do not dilute the stain beyond 1 200 as this will result in reduced intensity and sensitivity e Do not re use the stain solution as this may result in a significant loss of sensitivity e During the process gel containers should be covered to exclude light and agitated gently on a mixer platform e The source of SDS used to prepare and run polyacrylamide gels can affect the background obtained on imaging Use high quality materials Certain commercially available premade running buffers may not be suitable particularly when using short fixation times 80 6429 60 AD 137 Table A Typical stain and gel processing solution volumes for the Deep Purple Total Protein Stain protocol Electrophoresis system Gel dimensions cm Stain volume ml Processing solution volume ml Ettan DALTsix 20x 26 x 0 1 500 1000 Ettan DALTtwelve 20 x 26 x 0 1 500 1000 miniVE 10 x 10 x 0 05 50 100 SE 260 10 x 10 x 0 05 50 100 SE 600 18 x 16 x 0 1 250 500 cr For optimal performance it is critical that the pH in the gel is raised before adding the stain solution If not high background or negative staining may be observed Therefore the prope
122. ehydration Solution at room temperature for 30 min Shake the bottle to dissolve the urea crystals Sample preparation 1 Prepare the protein extract in sample buffer containing reducing agents such as dithiothreitol DTT B mercaptoethanol or tris 2 carboxyethyl phosphine TCEP at a concentration of 20 mM Note Using cup application the sample solution may contain up to 1 mg of protein ml Using anodic paper bridge loading higher concentrations can be used Preparation of DeStreak Rehydration Solution 1 DeStreak Rehydration Solution is supplied without IPG Buffer Before use add 15 pl 0 5 or 60 pl 2 0 of the appropriate IPG Buffer or Pharmalyte to 3 ml of DeStreak Rehydration Solution eve Use 0 5 IPG Buffer in the DeStreak Rehydration Solution when e PGphor standard Strip Holder is used for the first dimension 80 6429 60 AD 55 e Horizontal gels are used in the second dimension e 10 kV is used in the Manifold e Immobiline DryStrip 7 11 NL and 3 11 NL are used This will give a high voltage and a short run time in hours both of which are essential for streak free results Use 2 0 IPG Buffer in the DeStreak Rehydration Solution in cases where the highest solubility of proteins and stability against salt are needed Under these conditions conductivity will be higher and the highest voltage may not be reached Note Select an IPG Buffer with the same pH interval as the Immobiline DryStrip being rehydrate
123. ehydration buffer can lead o electroendosmosis which could dry out part of the strip possibly leading to arcing and burning in this region 68 80 6429 60 AD Table 21 Troubleshooting first dimension IEF Employing the Manifold Symptom Possible cause Remedy Current is too low or zero Electrical continuity is impeded Check the external electrode contacts Ensure correct placement of the electrode assemblies such that there is metal to metal contact with the appropriate electrode contact area Check the internal electrode contacts The gel which becomes visible because of the dye in the rehydration solution must contact both electrodes in the Manifold through the paper wicks and or paper bridge parts Check that the IPG strip is fully rehydrated along its entire length Electrical contact at the electrodes is reduced by incomplete rehydration Check that the paper wicks are present and properly positioned Voltage too low or does not reach maximum set value Ettan IPGphor protocol settings are incorrect for the experiment Check that the current limit is properly set Check that the actual number of Immobiline DryStrip gels on the Ettan IPGphor platform is the same as the number of gels entered in the protocol Conductivity ionic strength is too high Prepare the sample to yield a salt concentration less than 10 mM The recommended IPG Buffer concentration is 0 5 A maximum of 2 is ad
124. el buffer SOLUTION e ceccccccsssssssssssssstssesssssssesssseceeeseeeesssssssssssuummsssessesesseeeeesecsesssssssssnenee 131 J Bromophenol HIUS StOCK SOLUTION ssscscscaccszcrescadsesrectesenssassesasecsaeacassusseactucscasndseastsadasvshcctertsatartucstervacctcties 131 W10 SDS SOLION Serepan ETE E E ENE EEE 131 K 10 ammonium persulfate SOLILIOM ccscersscscessoreicsesccedsercsacsncscacscssessnscscasndvuasevdaadacsacsasvaaciactuchioesustinsies 132 l Gel stordge Solutio N cecacarcecsssvsatessusacoossassavieranooccvtnsscvisvesadasubussebaetasadsesceeshonaneshtoisesbosusasuanaseieasnsaz vactebetandasuue 132 M 1x Laemmli SDS electrophoresis buffer essssssssscccceccecesssesssssnsnumnnsessesesseceecseesessssssssansnes 132 N Agarose sealing SolUtION woe ecccsssssssssscccccsssssssssssnssusssscesessssesceeseesessssssssssusmunssssssecseseeeeeeeceesessssssntsnte 132 Appendik Mh cceucsscesseesececassvevscceteuecyccegtecuebcecesuetcnscevstsutscbensdshescesencesxsconsnuvascuedcucssesbteacecscscaselasbedodasenasavastsstenee 133 Optimized silver staining of large format DALT gels and DALT 12 5 precast gels using PlusOne Silver Staining Kit Protein esses 133 Appendix IN cicesscciscsssvscvscescscceatecanescdecivesasctancsanvescsstvais scabies nendtassennd eesoesai sate Esa raara EE NESSA E ERST 135 Colloidal Coornassie staining Procedure is scscrccszcsssasisssaiisssensvatsvcavecsesssassessensnuos count aseantaadasssaceasvasciasbactistsurssattin 135 5
125. elow Multiphor II Electrophoresis System can be used for both first dimension IEF and second dimension SDS PAGE Strip rehydration with or without sample included is performed in the Immobiline DryStrip IPGbox After rehydration the Immobiline DryStrip gels are transferred to the electrophoresis unit for first dimension IEF using the Immobiline DryStrip Kit accessory 4 1 First dimension IEF using Multiphor II Electro phoresis System and Immobiline DryStrip Kit Multiphor II Electrophoresis System can be readily configured for first dimension IEF separations by incorporating an Immobiline DryStrip Kit and choosing the Immobiline DryStrip gel and IPG Buffer to match the required pH gradient When equipped with Immobiline DryStrip Kit the system can run up to 12 Immobiline DryStrip gels simultaneously with gel lengths up to 24 cm Focusing time depends on the gel length pH range and the nature of the sample but can be expected to be in the range of 2 72 h DryStrip gels are rehydrated using a Reswelling Tray accessory in a solution containing the necessary additives and optionally the sample proteins rehydration solution is described in detail in section 2 6 IEF is performed by gradually increasing the voltage across the Immobiline DryStrip gels to at least 3500 V and maintaining this voltage for at least several thousand Volt hours After IEF the Immobiline DryStrip gels are equilibrated in equilibration solution and applied onto SDS polyac
126. els and related equipment for IEF and 2 D electrophoresis see chapters 2 and 3 For more specific guidance on preparing samples for application to Immobiline DryStrip gels see references 14 16 80 6429 60 AD 19 1 1 Methods of cell disruption Tables 5 and 6 list some standard mechanical and chemical disruption methods Cell disruption should be performed at low temperature keep the sample on ice as much as possible and use chilled solutions Proteases may be liberated upon cell disruption thus the protein sample should be protected from proteolysis if one of the methods described in this section is to be used It is generally preferable to disrupt the sample material directly into a strongly denaturing lysis solution to rapidly inactivate proteases and other enzymatic activities that may modify proteins Cell disruption is often carried out in an appropriate solubilization solution for the proteins of interest see references 17 and 18 for general information on tissue disruption and cell lysis 1 1 1 Gentle lysis methods Gentle lysis methods are generally employed when the sample of interest consists of easily lysed cells such as tissue culture cells blood cells and some microorganisms Gentle lysis methods can also be employed when only one particular subcellular fraction is to be analyzed For example conditions can be chosen in which only cytoplasmic proteins are released or intact mitochondria or other organelles are recovered by di
127. els are equilibrating as long as the timeframes above are adhered to 3 2 Background to SDS PAGE SDS PAGE is an electrophoretic method for separating polypeptides according to their molecular weights M The technique is performed in polyacrylamide gels containing sodium dodecyl sulfate SDS The intrinsic electrical charge of the sample proteins is not a factor in the separation due to the presence of SDS in the sample and the gel SDS is an anionic detergent that when in solution in water forms globular micelles composed of 70 80 molecules with the dodecyl hydrocarbon moiety in the core and the sulfate head groups in the hydrophilic shell SDS and proteins form complexes with a necklace like structure composed of protein decorated micelles connected by short flexible polypeptide segments 77 The result of the necklace structure is that large amounts of SDS are incorporated in the SDS protein complex in a ratio of approximately 1 4 g SDS g protein SDS masks the charge of the proteins themselves and the formed anionic complexes have a roughly constant net negative charge per unit mass Besides SDS a reducing agent such as DTT is also added to break any disulfide bonds present in the proteins When proteins are treated with both SDS and a reducing agent the degree of electrophoretic separation within a polyacrylamide gel depends largely on the molecular weight of the protein In fact there is an approximately linear relationship between the logar
128. ely at the wavelengths used for scanning Protocol to treat glass plates with Bind Silane v The glass plates must be properly cleaned Before re use soak the plates overnight in a 5 Decon 90 solu tion Do not leave plates standing in a Decon solution for a longer time as this will eventually cause etching due to the alkali nature of Decon 1 Thoroughly wash each plate to be treated Take care to remove any gel fragments attached to the plate from previous gels The careful cleaning of the glass plates before casting is important to ensure a uniform coating with the Bind Silane and to avoid keratin contamination 2 Thoroughly rinse the plates with double distilled water to remove Decon 3 Dry the plates using a lint free tissue or leave them to air dry 4 Prepare the Bind Silane working solution Ethanol 8ml Glacial acetic acid 200 ul Bind Silane 10 ul Double distilled water 1 8 ml 5 Pipette 2 4 ml depending on plate size of the Bind Silane working solution onto each plate and distribute equally over the plate with a lint free tissue such as Crew Wipes Cover the plate to prevent dust contamination and leave to air dry on the bench for 1 1 5 h 6 Polish the plate with a lint free tissue such as Crew Wipes moistened with a small amount of double distilled water or ethanol The gels will stay attached to the glass during electrophoresis staining procedures scanning and storage 80 6429 60 AD 141 142 80 6429 60 AD
129. ematic of the labeling reaction CyDye DIGE Fluor containing NHS ester active group covalently binds to lysine residue of protein via an amide linkage Minimal labeling of proteins does not affect the mass spectrometry data used to identify proteins because only 1 2 of lysine residues are labeled such that 98 of protein is unlabeled 6 2 CyDye DIGE Fluor labeling kits with saturation dyes for labeling scarce samples and preparative gels Two labeling kits for scarce samples are available one contains the Cy3 and Cy5 saturation dyes and the other contains an additional vial of Cy3 dye to label a preparative gel Each kit contains sufficient dye for at least 12 labeling reactions and allows labeling of as little as 5 ug of protein per labeling reaction compared with 50 ug with the minimal dyes The additional vial of Cy3 dye contained in the CyDye DIGE Fluor Labeling Kit for Scarce Samples and Preparative Gel Labeling allows for labeling up to 500 ug of protein The saturation dyes Cy3 and Cy5 retain the advantages described in section 6 1 for the minimal dyes The maleimide reactive group of the saturation dyes covalently bonds to the thiol group of cysteine residues of proteins via a thioether linkage To achieve maximal labeling of cysteine residues the protocol uses a high fluor to protein labeling ratio This type of labeling method labels all available cysteines on each protein under the conditions used resulting in the majority of cysteine
130. ems come above the sealing assembly when the electro phoresis unit is fully loaded If excess anode buffer is in the upper reservoir it should be removed with a pipette Ensure that the level of cathode buffer does not come above the air vents in the corners of the upper reservoir Lack of mixing between upper and lower reservoirs can be verified by adding bromophenol blue dye to he lower reservoir prior to loading the unit with gels Several drops of 1 w v bromophenol blue will impart sufficient color to the anode buffer Incomplete gel Chemicals Use only fresh stocks of the highest quality reagents polymerization f the dry ammonium persulfate does not crackle when added to water replace it with fresh stock ncrease TEMED or ammonium persulfate concentration or both Oxygen Remove oxygen from the gel environment Degas he monomer solution 5 10 min before pouring and hen overlay the gel surface with water saturated 1 butanol Temperature Adjust the gel solution temperature to a minimum of 20 C especially for gels with low acrylamide concentration Pronounced downward There is an unfilled gap between the When sealing the Immobiline DryStrip gel into place curving of the dye front gel and one of the spacers on top of the gel ensure that some of the sealing on one or both sides of solution flows down any gap that may exist between the DALT Gel 12 5 the gel and spacer Precast gel cassette s not E
131. ension IEF Ettan IPGphor 3 Isoelectric Focusing System e Table 21 page 71 Troubleshooting first dimension IEF Employing the Manifold e Table 34 page 90 Troubleshooting vertical second dimension SDS PAGE e Table 36 page 95 Troubleshooting Immobiline DryStrip gel rehydration in Reswelling Tray e Table 38 page 103 Troubleshooting first dimension IEF Multiphor II Electrophoresis System and Immobiline DryStrip Kit e Table 40 page 107 Troubleshooting second dimension SDS Multiphor II Electrophoresis System For troubleshooting 2 D DIGE results please refer to the Ettan DIGE user manual Table 44 Troubleshooting 2 D results Symptom Possible cause Remedy No distinct spots are visible Individual proteins appear as multiple spots or are missing unclear or in the wrong position Sample is insufficient nsufficient sample entered he Immobiline DryStrip gel due to poor sample solubilization Increase the amount of sample applied Increase the concentration of the solubilizing components in the sample solution see section 1 6 Sample contains impurities hat prevent focusing Increase the focusing time or modify the sample preparation method see chapter 1 The pH gradient is incorrectly oriented The end of the Immobiline DryStrip is the acidic end and should point toward the anode Flatbed gel format Immobiline DryStrip gel is placed wrong side down
132. ent stain that provides e High sensitivity e Clear easily discernible and accurately quantitated protein spots and bands e High signal to noise ratios so low intensity spots and bands are detected e Compatibility with most fluorescent scanners and CCD cameras UV transilluminators and some light boxes Ease of disposal and environmental friendliness naturally occurring fluorophore is free from heavy metals e Low viscosity and thus easy to handle with no oily residue 80 6429 60 AD 105 Deep Purple Total Protein Stain is compatible with downstream analysis such as MS and Edman sequencing and is ideal for post staining gels used in 2 D DIGE analysis with Ettan DIGE system see chapter 6 Alternatives to Deep Purple Total Protein Stain include Sypro dyes 87 90 which have a sensitivity between colloidal Coomassie and modified staining using PlusOne Silver Staining Kit Protein 84 Deep Purple Total Protein Stain provides superior 1 D and 2 D gel image data compared with Sypro Ruby dyes and clearer backgrounds see additional reading and reference material Refer to appendix IV for the protocol for use of Deep Purple Total Protein Stain Figure 57 shows 2 D gels stained with Sypro Ruby and Deep Purple Total Protein Stain Fig 57 2 D gels of a protein sample consisting of a mix of HBL100 breast cell line and BT474 breast cell carcinoma stained with A Sypro Ruby and B Deep Pur
133. entially more salt buffer carryover This water movement is also more pronounced when working with basic IPG strips pH ranges 6 9 6 11 and 7 11 NL Moistened prior to use the paper wicks have the ability to absorb excess ions and buffers that move to the electrodes and that may otherwise perturb the focusing They also serve to absorb the water accumulating at the cathodic side of the strip as H O and to keep the anodic side of basic strips hydrated potentially they can dry out from the depletion of water Moreover since preparative loads are usually applied to narrow range strips the wicks will soak up the excess proteins that lie above and below the pH range being studied It is easy to apply a cup to the surface of the gel for sample application Cup loading can be advantageous for basic proteins and also for very acidic proteins mostly due to stability issues with these proteins once they reach their pl Sharper spots can be obtained by loading samples away from their pl Thus basic proteins would be loaded at the anodic side and acidic proteins at the cathodic side e The electrodes are fully adjustable to suit the length of strip The Manifold can be used to run any IPG strip with a length between 7 and 24 cm One to 12 strips all of the same length can be run at the same time e No pressure is exerted against the strip surface Because the gel is run face side up there is no pressure of the gel against the ceramic surface suc
134. erefore minimizing intermolecular interactions See section 1 6 for a discussion of the components of sample preparation solutions The original method for first dimension IEF depended on carrier ampholyte generated pH gradients in cylindrical polyacrylamide gels cast in glass rods or tubes 1 Carrier ampholytes are small soluble amphoteric molecules with a high buffering capacity near their pl When a voltage is applied across a carrier ampholyte mixture the carrier ampholytes with the highest pl and the most negative charge move toward the anode those with the lowest pl and the most positive charge move toward the cathode The other carrier ampholytes align themselves between the extremes according to their pls and buffer their environment to the corresponding pHs The result is a continuous pH gradient As a result of limitations and problems associated with carrier ampholyte pH gradients immobilized pH gradients IPG were developed GE Healthcare subsequently introduced Immobiline chemicals for the generation of this type of pH gradient 2 Gorg et al 3 4 pioneered the development and use of IPG IEF for the first dimension of 2 D electrophoresis An immobilized pH gradient is created by covalently incorporating a gradient of acidic and basic buffering groups immobilines into a polyacrylamide gel at the time it is cast Immobiline buffers are a set of well characterized molecules each with a single acidic or basic buffering group
135. eseseeeeeeseessssssssuannees 84 3 4 2 Preparing lab cast gels for miniVE SE 260 and SE 600 Ruby electrophoresis SYStEMS ccccccccssssssccccsssssssssssssssuussssssesseseseeeeeeseessssssssnanee 84 3 4 3 Preparing miniVE SE 260 and SE 600 Ruby systems for electrophoresis 86 3 4 4 Equilibrating IMMObILING DryStrip GEIS ccecececccccsccccssssssesssssssssnsssesseceeeceeceeseessessssssssunsesess 86 3 4 5 Applying Immobiline DryStrip gels s87 3 4 6 Inserting gels into miniVE SE 260 and SE 600 Ruby SyYStemMS s s 1 s11511511151151151111 87 3 4 7 Electrophoresis COnditions cssssssssssssssesssssessesececcecceeseessssssssnssuunssssseseeseeceeeceeesessssssassnssnnuueee 87 3S MOUBlESNOOTN sesin i E Aa A AR 88 4 Use of the flatbed Multiphor II Electrophoresis System for first and second dimensions 91 EV TOYS OWN zich titan catecsceectacato a a EN E E AAAA REE 91 4 1 First dimension IEF using Multiphor II Electrophoresis System and Immobilie Dry Strip KiE issnin nsaisan ee EE E EEEE 91 4 1 1 Immobiline DryStrip gel rehydration IPGbOX eeeeeessssssssssssscsceccccessessssssnsnnsnnusssssesseseeeeeeeees 92 GLZ PRE BERING MOR EP sis ecsicchsscsczetassstessastecceranssscesset Peantenests dood ede AR Wes aasanchee 93 4 1 3 Sample application by CUP loading ncceeecccscssssssssssssssssssusssssssssssssseeeesesssssssssnsssunusssssesseseeeeeeeees 94 GVA Paper bridge lOdding sssssssssssssssssssss
136. ette Inserts into any unoccupied slots 3 When all 12 slots are occupied the lower buffer level should be slightly below the level of the gaskets Pour the diluted 1x cathode buffer into the upper portion of the tank to the fill line some of this buffer may drip through the gasket and mix with the anode buffer during the run but this will not affect performance or results 4 Close the lid Fig 44 Loading the gel cassettes into Ettan DALTtwelve electrophoresis unit 80 6429 60 AD 79 3 3 6 Electrophoresis conditions with precast gels for both Ettan DALTsix and Ettan DALTtwelve Table 23 lists the recommended conditions for Ettan DALTsix and Ettan DALTtwelve systems Electrophoresis is performed at constant power in two steps Stop electrophoresis when the dye front is approximately 1 mm from the bottom of the gel Temperature control improves gel to gel reproducibility especially if the ambient temperature of the laboratory fluctuates significantly For best results gels should be run at 25 C After electrophoresis remove gels from their gel cassettes in preparation for staining or blotting Precast gels have a barcode number and gel percentage printed on them which should be noted for orientation Table 23 Recommended electrophoresis conditions for second dimension vertical gels Step Power Approximate run W gel duration h min Ettan DALTsix set temperature to 25 C 1 mm thic
137. extraction Carrier ampholytes Pharmalytes Ampholines or IPG Buffers can be added at concentrations up to 2 for standard protocols but should not be added during protein extraction for labeling in 2 D DIGE 1 Allow the solution to warm to room temperature 2 Vortex briefly before using as the solution is in suspension form 3 Dilute Protease Inhibitor Mix 1 100 10 ul ml in an appropriate volume of extraction buffer or extract Further options e Ifa higher potency of protease inhibition is required add Protease Inhibitor Mix at a concentration of 20 30 yl ml to give a 2 3x final concentration e For the inhibition of metalloproteases add EDTA directly in an appropriate volume of extraction buffer or extract to give a final concentration of 5 mM EDTA in the reaction EDTA must not be added if the solution is to be used in conjunction with Nuclease Mix because EDTA acts as a nuclease inhibitor 1 3 Fractionation of protein lysates Proteome studies involving quantitative comparisons of total cell protein profiles from two or more experimental samples require methods for highly reproducible separation of cell or tissue protein extracts 2 D gel electrophoresis is currently the only proven method for simultaneous separation of highly complex protein mixtures and quantitative comparison of changes in protein profiles of cells tissues or whole organisms Although 2 D electrophoresis gives the highest resolution of all available protein
138. ffer the convenience of ready to use gels and buffer strips F The protein loading capacity of an Immobiline DryStrip gel can exceed the capacity of the thin horizontal second dimension gel so thicker vertical second dimension gels are preferred for micropreparative separations F The Multiphor Electrophoresis System is not recommended for the second dimension step if pH 6 9 6 11 or 7 11 NL Immobiline DryStrip gels have been used for the first dimension separation 14 80 6429 60 AD Good laboratory practice Always wear gloves when handling Immobiline DryStrip gels SDS polyacrylamide gels ExcelGel Buffer Strips and any equipment that these items will contact The use of gloves will reduce protein contamination that can produce spurious spots or bands in 2 D patterns Clean all assemblies that will be in contact with the gels or samples using a detergent designed for glassware and rinse well with distilled water This is particularly important when highly sensitive mass spectrometry techniques are employed for spot identification and characterization A special detergent is available for the Strip Holders and Manifold see chapter 2 ce Always use the highest quality reagents and the purest water available g Some of the chemicals used in the procedures acrylamide N N methylenebisacrylamide ammonium persulfate TEMED thiourea DTT iodoacetamide and DeStreak Reagent are very hazardous Acrylamide monomer for example
139. fferential centrifugation Sometimes these techniques are combined e g osmotic lysis following enzymatic treatment freeze thaw in the presence of detergent Table 5 summarizes various options for gentle lysis Table 5 Gentle lysis methods Cell disruption method Application General procedure Osmotic lysis 19 This very gentle method is well suited for Blood cells Suspend cells in a hypo osmotic solution applications in which the lysate is to be tissue culture subsequently fractionated into subcellular cells components Freeze thaw lysis 9 17 20 Many types of cells can be lysed by Bacterial cells Rapidly freeze cell suspension using subjecting them to one or more cycles tissue culture liquid nitrogen then thaw of quick freezing and subsequent thawing cells Repeat if necessary Detergent lysis 21 22 Detergents solubilize cellular membranes Tissue culture Suspend cells in lysis solution containing detergent lysing cells and liberating their contents cells Cells can often be lysed directly into sample solution or rehydration solution because these solutions always contain detergent See appendix solution A for an example of a widely used lysis solution Further examples of this technique are given in references 21 and 22 If an anionic detergent such as SDS is used for lysis one of the following preparation steps is required to ensure that the SDS will not interfere with IEF Dilute the lysed sa
140. fore raising the voltage to maximum include a prolonged low voltage phase in the IEF protocol to allow the ions to move to the ends of the Immobiline DryStrip gel 100 80 6429 60 AD 4 2 Second Dimension SDS PAGE using Multiphor II Electrophoresis System w As discussed in chapter 2 after IEF it is important to proceed immediately to gel equilibration unless the IPG strip is being frozen at 60 C or below for future analysis Equilibration is always performed immediately prior to the second dimension run never prior to storage of the Immobiline DryStrip gels See section 4 1 8 for details on preservation of the gels w The second dimension gel itself should be prepared and ready to accept the Immobiline DryStrip gel prior to equilibration Before proceeding further refer to sections 3 1 1 and 3 1 2 for a discussion of the equilibration process Note especially the discussion referring to the equilibration solution components and the need for a second equilibration step with iodoacetamide 4 2 1 ExcelGel preparation Two sizes of precast ExcelGel SDS gels are recommended for 2 D electrophoresis e ExcelGel SDS 2 D Homogeneous 12 5 11 x 25 cm e ExcelGel SDS Gradient XL 12 14 18 x 25 cm Both gels accept a single 24 18 or 13 cm Immobiline DryStrip gel two 11 cm or three 7 cm Immobiline DryStrip gels Placing shorter Immobiline DryStrip gels end to end is ideal for comparative studies For maximum resolution the larger gel c
141. fully peel back the foil on the yellow colored anodic strip and place it in the appropriate slot of the positioner For 11 x 25 cm ExcelGel SDS gels place the anodic strip in slot 3 in the center of the positioner For 18 x 25 cm ExcelGel SDS gels place the anodic strip in slot 4 anodic edge of the positioner Fr The buffer strips should sit snugly within the slots 4 2 2 Applying equilibrated Immobiline DryStrip gels Protocol 1 Drain moisture from Immobiline DryStrip gels flatbed second dimension only After equilibration place the Immobiline DryStrip gels on filter paper moistened with deionized water To help drain the equilibration solution place the Immobiline DryStrip gels so they rest on an edge They can be left in this position for up to 10 min without noticeably affecting the spot sharpness Alternatively the Immobiline DryStrip gels can be gently blotted with moistened filter paper to remove excess equilibration buffer 2 Position the lmmobiline DryStrip gel s Once the equilibrated Immobiline DryStrip gels have drained for at least 3 min use forceps to place them gel side down on the ExcelGel through the slot at position 2 Fig 54 The anodic side of the IPG DryStrip should be oriented such that it is toward the front edge of the gel Fig 53 Positioning the anodic buffer strip on Multiphor II unit Fig 54 Positioning equilibrated Immobiline DryStrip gels on Multiphor II unit such that the anodic acidic side
142. g dialysis the tube is centrifuged briefly This forces the entire contents of the dialysis tube into the bottom of the tube ensuring essentially 100 recovery The dialyzing cap is replaced with a normal 32 80 6429 60 AD cap for storage of the dialyzed sample Fig 11 The kit is available with a choice of molecular weight cut off either 1 kDa or 8 kDa and a choice of tubes for sample volumes of either 250 ul or 2 ml Each kit contains dialysis tubes and associated accessories sufficient for preparing 50 samples Dialysis times of a few hours to overnight are sufficient to reduce ionic contaminants to a level that does not interfere with first dimension IEF separation Since some detergents notably Triton X 100 and SDS form high molecular weight micelles at low concentration they cannot be effectively removed by dialysis Other techniques such as sample precipitation with 2 D Clean Up Kit see section 1 4 1 must be used to remove these detergents Protocol Mini Dialysis Kit Components supplied Dialysis tubes with caps incorporating a dialysis membrane tubes for up to 250 ul or 2 ml sample are included in the kit caps standard tube caps to seal the tubes following dialysis floats floating plastic sponges to suspend the inverted dialysis tubes in the dialysis solution Required but not provided Centrifuge dependent on size of dialysis tube capable of low speeds Preliminary notes Prior to dialysis samples for native IE
143. g rehydration is a concern EF Active rehydration 20 120 V can also be performed if sample is included Fig 26 Applying rehydration solution into the Strip Holder a Positioning the Immobiline DryStrip gel b c Positioning the Immobiline DryStrip gel A Rehydration loading A discussion of the advantages of rehydration loading can be found in section 2 4 B Optional Apply electrode pads During IEF the transport of ions proteins and IPG Buffer between the electrodes is accompanied by transport of water For large sample loads and when using narrow pH range Immobiline DryStrip gels better results are obtained by applying damp paper pads between the Immobiline DryStrip gel and each Strip Holder electrode following rehydration but before IEF in order to absorb excess water 1 Prepare electrode pads Use the paper wicks accessory to the Manifold or cut two 3 mm wide electrode pads from a paper IEF electrode strip Place on a clean flat surface such as a glass plate and soak with deionized water Remove excess water by blotting with tissue paper mo Electrode pads must be damp not wet 2 Position electrode pads Using forceps lift one end of the rehydrated Immobiline DryStrip gel Position an electrode pad over the electrode then lower the gel strip back into place Repeat at the other end ele Additional DryStrip Cover Fluid may need to be added to ensure that the strip is still adequately covered 80 6429 60 AD 59
144. ges of using the flatbed format are e Since the pl of a protein is dependent on the temperature precise cooling is required during IEF This can be effectively achieved by using the aluminum oxide ceramic Strip Holder or Manifold in conjunction with a Peltier temperature controlled bed e Since IEF requires high field strengths to obtain sharply focused bands high voltages must be applied A flatbed design is the most economical way to meet the necessary safety standards required to operate at such high voltages 2 2 Immobiline DryStrip gels Immobiline DryStrip gels offer a marked improvement over tube gels using carrier ampholyte generated pH gradients When Immobiline DryStrip gels are used for the first dimension separation the resultant 2 D spot maps provide superior results in terms of resolution and reproducibility e The first dimension separation is more reproducible because the covalently fixed gradient cannot drift e Plastic backed Immobiline DryStrip gels are easy to handle They can be picked up at either end with forceps or gloved fingers e The plastic support film prevents the gels from stretching or breaking e IPG technology increases the useful pH range on any single Immobiline DryStrip gel more very acidic and basic proteins can be separated e Immobiline DryStrip gels have a higher protein loading capacity 69 e The sample can be introduced into the Immobiline DryStrip gel during rehydration 70 71 e Precas
145. gy which lacks selectivity and can remove low abundance proteins of interest See Figures 12 and 13 for typical results Figure 14 depicts the methodology used in the kit 34 80 6429 60 AD 1 2 3 4 5 6 7 8 9 10 Lane 1 Human Serum Albumin _ Lane 2 Human IgG Lane 3 Untreated serum 5 oo ee ES ee SE Geer Lane 4 Treated serum replicate 1 Lane 5 Untreated serum Lane 6 Treated serum replicate 2 _ _ lt Lane 7 Untreated serum Lane 8 Treated serum replicate 3 Lane 9 Untreated serum _ Lane 10 Treated serum replicate 4 Fig 12 Typical results when using the Albumin and IgG Removal Kit Four replicates of a human serum sample were treated with Albumin and IgG Removal Kit using the standard protocol untreated samples in lanes 3 5 7 and 9 and treated samples in lanes 4 6 8 and 10 Untreated human serum was diluted to an equivalent volume Equivalent amounts of untreated and treated serum were separated onto a 12 acrylamide gel alongside purified albumin and IgG The gel was stained with Sypro Ruby and imaged on Typhoon 9400 Variable Mode Imager Untreated human serum Human serum treated with Albumin and IgG Removal Kit Dooi oe DOn T Tk gt er AR I an gt o A Fig 13 Removal of albumin from human serum Following treatment with Albumin and IgG Removal Kit albumin is removed and lower abundance proteins gain increased resolution The albumin region of t
146. h as in the regular Strip Holder This is advantageous when running preparative loads as it lessens the streaking associated with abundant proteins There are however several drawbacks to running strips gel side up the inability to apply voltage during rehydration the extra manipulation of the strip from tray to Manifold and the absence of active temperature control during the rehydration step Er For best results with the basic IPG strips in addition to anodic cup loading the use of DeStreak Reagent is highly recommended see section 2 6 2 The Manifold tray base is made of a thermally conductive aluminum oxide ceramic that rapidly dissipates heat to avoid potential hot spots A further special coating of the surface eliminates protein absorption The Manifold tray allows simple and accurate placement of IPG strips with protrusions along the numbered inner channels that keep IPG strips straight and centered Pre cut electrode pads and paper bridges are convenient and save valuable time The wicks absorb excess water salts and proteins while the paper bridges can be used to load large sample volumes The overall procedure for use of Ettan IPGphor 3 with the Manifold is depicted in Figure 23 Following sections provide protocols for use of the various accessories Wi After IEF proceed to the second dimension separation immediately or store the Immobiline DryStrip gels at 60 C or below as described in section 2 8 3 The Etta
147. hand while pouring the solution with the other Fig 35 3 Switch on the separation unit 4 Turn on the pump to mix the buffers and set the separation unit to desired temperature ae A temperature of 25 C is recommended for electrophoresis re a 2 Fig 35 Spreading the tubing elements apart with one hand while pouring the solution with the other to avoid pouring the 100x anode buffer onto the tubing 80 6429 60 AD 75 3 3 2 Inserting DALT Gel 12 5 into DALT Precast Gel Cassette Protocol 1 Open the gel package Cut around the package on two sides at approximately 1 cm from the edge to avoid cutting the gel or the support film Remove the gel from the package The gel is cast onto a plastic support film and does not cover the film entirely The gel is covered with a protective plastic sheet Markings on the protective sheet indicate the orientation of the gel and the direction of electrophoresis The bottom or anodic edge of the gel is flush with the edge of the support film The support film protrudes approximately 15 mm beyond the top or cathodic edge of the gel and approximately 5 mm at either side 2 Open DALT Precast Gel Cassette Place the gel cassette on the bench top with the hinge down Apply 1 ml gel buffer onto the glass plate as a streak along the spacer on the right edge of the glass plate Fig 36 Add an additional 2 ml of gel buffer to the center of the plate Fig 36 Pipetting a streak of gel buffer
148. he anode Fig 19 Two dimensional electrophoresis of mouse liver extract with or without alkylated lysozyme using broad and medium range 24 cm Immobiline DryStrip gels All first dimension results shown in Figure 19 were run on Ettan IPGphor 3 with Ettan IPGphor 3 Manifold Immobiline DryStrip gels were rehydrated in DeStreak Rehydration Solution with IPG Buffer solutions as indicated Second dimension Ettan DALT twelve using DALT Gel 12 5 precast SDS PAGE gels Staining PlusOne Silver Staining Kit Protein White rings same protein seen on two pH intervals i e overlaps between pH gradients Rings or ovals of the same color present on the broad pH gradient 3 11 NL Fig 19a and the medium range gradients Figs 19b 19e indicate the same protein groups Yellow ring alkylated lysozyme pl 10 5 calculated according to SwissProt 2 2 3 Choosing an IPG Buffer IPG Buffers are ampholyte containing buffer concentrates specifically formulated for use with Immobiline DryStrip gels Each IPG Buffer type produces more uniform conductivity along the Immobiline DryStrip during focusing resulting in wider latitude in run times IPG Buffers also eliminate potential high background staining The buffers supplied in 1 ml aliquots are diluted 50 or 200 fold in the rehydration solution depending on the first dimension system and pH range of the strip Figure 20 shows the appropriate IPG Buffer for use with the various IPG DryStrip gels 2 2 4 Estimating
149. he gel before and after treatment is shown above Pan Discard upper chamber Incubate Transfer slurry Concentrate desalt serum resin with to upper sample as required mixing chamber of MicroSpin column Fig 14 Schematic of the removal process Optimal albumin and IgG binding gt 95 total protein is achieved using a 15 yl human serum loading and will typically lead to recovery of between 150 and 220 ug of lower abundance proteins The amount of protein recovered will vary with the protein content of the individual serum samples used Protocol Albumin and IgG Removal Kit Components supplied 8 5 ml of a 50 50 v v resin slurry 10 empty microspin columns 10 microcentrifuge tubes and lids wy Some of the components contain sodium azide in dilute solution This substance is classified as toxic when undiluted Follow all local safety regulations when disposing of waste Unless local regulations dictate other methods dispose of waste by flushing with copious amounts of water to avoid the build up of explosive metallic azides in copper and lead plumbing Required but not provided Microcentrifuge rotary shaker disposable 15 ml centrifuge tube If performing acetone precipitation for concentration desalting acetone and acetone resistant microcentrifuge tubes are also required Acetone precipitation is not necessary if using the 2 D Clean Up Kit Preliminary notes Ensure that the resin is in suspension when removing resin aliquots Vigor
150. hey apply to vertical systems sections 4 1 and 4 2 describe them as they apply to the flatbed Multiphor II Electrophoresis System 3 1 Equilibrating Immobiline DryStrip gels w As mentioned in chapter 2 after IEF it is important to proceed immediately to gel equilibration unless the IPG strip is being frozen at 60 C or below for future analysis Equilibration is always performed immediately prior to the second dimension run never before storage of the Immobiline DryStrip gels The second dimension gel itself should be prepared and ready to accept the Immobiline DryStrip gel before beginning the equilibration protocol 3 1 1 Equilibration solution components The equilibration step saturates the Immobiline DryStrip gel with the SDS buffer system required for the second dimension separation The equilibration solution contains buffer urea glycerol reductant SDS and dye An additional equilibration step replaces the reductant with iodoacetamide Equilibration buffer 75 mM Tris HCl pH 8 8 maintains the Immobiline DryStrip gel in a pH range appropriate for electrophoresis Urea 6 M together with glycerol reduces the effects of electroendosmosis by increasing the viscosity of the buffer 3 Electroendosmosis is due to the presence of fixed charges on the Immobiline DryStrip gel in the electric field and can interfere with protein transfer from the Immobiline DryStrip gel to the second dimension gel Glycerol 30 together with
151. hlighting use of Ettan IPGphor 3 Isoelectric Focusing System Chapter 3 contains general directions for subsequent second dimension electrophoresis of immobilized pH gradient IPG strips using various vertical gel electrophoresis systems Chapter 4 describes use of the flatbed Multiphor II Electrophoresis System for both first and second dimension electrophoresis Chapter 5 discusses visualization and analysis of 2 D electrophoresis results Chapter 6 describes the advantages and use of the technique of 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE Chapter 7 describes common problems in 2 D gel electrophoresis and their remedies Technique specific troubleshooting guides are included in the relevant chapters The protocols described in this handbook are performed using products from Amersham Biosciences now a part of GE Healthcare referred to hereafter as GE Healthcare Equipment choices are illustrated in Table 1 Product ordering information is given on page 155 Depending on the sample type and the nature of the investigation the procedures may need to be adjusted or optimized Introduction to 2 D electrophoresis 2 D electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells tissues or other biological samples This technique separates proteins according to two independent properties in two discrete steps The first dimension step isoelectric focu
152. horesis with optimized systems First dimension IEF using Ettan IPGphor 3 Isoelectric Focusing System Gel sizes are given as gel width x separation length Ettan IPGphor 3 Isoelectric Focusing System with Ettan IPGphor Manifold or Standard Strip Holder Note The original IPGphor is fully compatible with the Manifold and with the protocols described throughout this handbook Fig 1 Ettan IPGphor 3 Isoelectric Focusing System Choice Factors Unique design of Ettan IPGphor 3 Manifold allows IEF of up to 12 IPG strips from 7 to 24 cm in length with subsequent equilibration of the strips Protein focusing patterns can be improved using cup based sample application particularly in basic IPG strips Manifold tray base is made of a thermally conductive aluminum oxide ceramic that rapidly dissipates heat to avoid hot spots Voltage current temperature and time controls are programmable Integral control software with an external personal computer Windows connected via a serial port can be used to control up to four Ettan IPGphor 3 units simultaneously each running a different set of run parameters Up to 10 protocols nine steps each can be saved retrieved and easily edited on the instrument Second dimension SDS PAGE using various vertical electrophoresis systems Ettan DALTsix Large Vertical System up to six 26 x 20 cm gels Fig 2 Ettan DALTsix Large Vertical System Choice Factors Four hou
153. ife span of the IPGbox Insert Protocol Preparing the Manifold 1 Clean and dry the IPGphor 3 bed before placing the Manifold tray on the unit Position the Manifold on the IPGphor 3 platform The small T shaped protrusion fits into a cutout section of the IPGphor bed near the lid hinge Fig 29 Ensure that the Manifold is level by placing the round spirit level on the center of the Manifold tray after it is placed on the Ettan IPGphor 3 unit Adjust leveling feet if necessary W Important Before proceeding make sure the Ettan IPGphor 3 unit is placed on a level surface Important If using the original Ettan IPGphor ensure that the three foam pads have been removed from the lid of the unit This step is not necessary if using Ettan IPGphor 3 Measure out 108 ml of Immobiline DryStrip Cover Fluid even if fewer than 12 strips will be loaded into the Manifold Add the cover fluid evenly between the 12 Manifold channels Transfer the strips to the Ettan IPGphor Manifold Place the strips under the cover fluid gel side up in the tray with the anodic end of the IPG strip oriented toward the anodic side of the instrument Position the strip to rest on the appropriate mark etched into the bottom of the Manifold channel the end of the gel not the end of the plastic should align with the etched mark Center the strip down the length of the Manifold channel Protrusions along the sides guide the strip approximately straight although som
154. ify the spot pattern by reducing the number of spots caused by protein oxidation compare A and B in Figure 25 When rehydrating Immobiline DryStrip gels with solutions other than DeStreak Rehydration Solution DeStreak Reagent can be added to the sample solution to stabilize thiol groups and prevent nonspecific oxidation DeStreak Reagent is compatible with most sample solutions as long as they do not contain more than 20 mM reducing agents such as dithiothreitol DTT B mercaptoethanol or tris 2 carboxyethyl phosphine TCEP Fig 25 A Without DeStreak Rehydration Solution Anodic cup loading Sample 100 ul 0 8 mg ml mouse liver protein contained 8 M urea 0 5 CHAPS 1 Pharmalyte pH 8 10 5 and 10 mM DTT Immobiline DryStrip gel pH 6 9 24 cm rehydrated in 1 IPG Buffer pH 6 11 with 8 M urea 0 5 CHAPS and 10 mM DTT B With DeStreak Rehydration Solution Anodic cup loading Sample 100 ul 0 8 mg ml mouse liver protein contained 8 M urea 0 5 CHAPS 1 Pharmalyte pH 8 10 5 and 10 mM DTT Immobiline DryStrip gel pH 6 9 24 cm rehydrated in DeStreak Rehydration Solution and 1 IPG Buffer pH 6 11 Protocol DeStreak Rehydration Solution Reagents supplied DeStreak Rehydration Solution 5 x 3 ml Required but not provided Sample buffer containing reducing agents up to 20 mM such as DTT B mercaptoethanol or tris 2 carboxy ethyl phosphine TCEP IPG Buffer or Pharmalyte Preliminary steps Before use equilibrate DeStreak R
155. ilm Alternatively the gel cassettes can be stored fully immersed in gel storage solution For further information on the preparation of second dimension vertical SDS slab gels refer to the user manuals for the respective vertical gel unit and multiple gel caster Table 26 Single percentage gel recipes for Ettan DALT systems Final gel concentration 10 12 5 15 Monomer solution solution G 300 ml 375 ml 450 ml 4x resolving gel buffer solution H 225 ml 225 ml 225 ml 10 SDS solution J 9 ml 9ml 9ml Double distilled water 360 7 ml 285 7 ml 210 8 ml 10 ammonium persulfatet solution K 5 ml 5ml 5ml TEMEDt 0 30 ml 0 25 ml 0 20 ml Total volume 900 ml 900 ml 900 ml Preparation of stock solutions is described in appendix solutions G H J and K Adjust as necessary for the thickness of the gels and the number of gels cast t Add after optional deaeration Table 27 Recipes for gradient gels for Ettan DALT systems Light solution Final concentration 8 10 12 14 16 Monomer solution solution G 120 ml 150 ml 180 ml 210 ml 240 ml 4x resolving gel buffer solution H 113 ml 113 ml 113 ml 113 ml 113 ml 10 SDS solution J 4 5 ml 4 5 ml 4 5 ml 4 5 ml 4 5 ml Double distilled water 210 5 ml 180 5 ml 150 5 ml 120 5 ml 90 5 ml 10 ammonium persulfatet solution K 18 ml 18 ml 1 8 ml 18 ml 18 ml TEMEDt 0 225 ml 0 225 ml 0 225 ml 0 225 ml 0 225 ml Total volume 450 ml 450 ml 450 ml 450 ml 450
156. in batches of up to 12 gels at a time DALT gels can also be poured using the DALT gel caster DALT gels can also be run using the DIGE buffer kit Detailed protocols for gel casting can be found in the Ettan DIGE user manual Ettan DALTtwelve user manual and Ettan DALTsix user manual The procedures for equilibrating strips positioning them and electrophoresis are identical to those for standard 2 D analysis Refer to sections 3 1 and 3 3 for details cr If the gels are to be scanned immediately store the gels in SDS electrophoresis running buffer at room temperature in a light tight container Scan the gels as soon as possible as the protein spots in the gel will diffuse with time If the gels cannot be scanned on the day of running they should be stored in the dark at 4 C and kept moist Remember to let the gels warm to room temperature before scanning because temperature affects the fluorescent signal Py Do not fix the gels prior to scanning as this will affect quantitation of the labeled protein spots 80 6429 60 AD 121 6 3 5 Summary of key differences between minimal labeling and saturation labeling Table 43 lists the key differences between minimal labeling and saturation labeling Table 43 Comparison of minimal labeling and saturation labeling experiments Saturation labeling Minimal labeling Sample preparation Cell lysis buffer is at pH 8 0 Cell lysis buffer is at pH 8 5 Dyes Maleimide dyes La
157. in chilled French pressure cell from forcing suspension through a small with cell walls Apply pressure and collect extruded lysate orifice under high pressure bacteria algae yeasts Grinding 5 8 28 29 Some cell types can be lysed by grinding Solid tissues Tissue or cells are normally frozen with liquid with a mortar and pestle microorganisms nitrogen and ground to a fine powder Alumina Al O or sand may aid grinding Mechanical homogenization 9 19 30 32 Many different devices can be used to Solid tissues Chop tissue into small pieces if necessary mechanically homogenize tissues Add chilled homogenization buffer 5 20 volumes Hand held devices such as Dounce or to volume of tissue Homogenize briefly Potter Elvehjem homogenizers can be used Clarify lysate by filtration and or centrifugation o disrupt cell suspensions or relatively soft issues Blenders or other motorized devices can be used for larger samples Homogenization is rapid and causes little damage to proteins except from the proteases hat may be liberated upon disruption Glass bead homogenization 23 24 33 The abrasive action of the vortexed Cell suspensions Suspend cells in an equal volume of chilled lysis beads breaks cell walls liberating the microorganisms solution and place into a sturdy tube Add 1 3 g cellular contents of chilled glass beads per gram of wet cells Vortex for 1 min and incubate cells on ice 1 min Repeat vortexing
158. ine DryStrip gel resulting in a lower final voltage when the system is limited by the maximum current setting Cr Longer focusing times may therefore be required at IPG Buffer Pharmalyte concentrations higher than 0 5 cr For higher protein loads up to 1 mg or more the final focusing step of each protocol can be extended if necessary by an additional 20 of the total recommended Volt hour value Exceeding the current limit of 50 uA Immobiline DryStrip gel is not recommended as this may result in excessive heat generation and may damage the Immobiline DryStrip gel and or Manifold or Strip Holder Under extreme circumstances the Immobiline DryStrip gel may burn ty Overfocusing can sometimes occur on longer runs and may contribute to horizontal streaking which will be visible in the 2 D gel result see also chapter 7 Troubleshooting 2 8 1 Protocol examples Ettan IPGphor 3 Isoelectric Focusing System These protocols are suitable for first dimension isoelectric focusing of protein samples prepared in rehydration solution in typical analytical quantities 1 100 ug The protocols are optimized for a rehydration solution containing 0 5 IPG Buffer or Pharmalyte The recommended current limit is 50 uA Immobiline DryStrip gel Recommended focusing times are given but the optimal length of time will depend on the nature of the sample the amount of protein and the method of sample application Please refer to the Ettan IPGphor 3 user manual
159. internal standard to be resolved on the same 2 D gel 4 Gel images are obtained from the Typhoon Variable Mode Imager which has been optimized for use with CyDye DIGE Fluor minimal dyes 5 The images are automatically analyzed within DeCyder 2 D Differential Analysis Software 6 112 80 6429 60 AD 6 1 CyDye DIGE Fluor dyes 6 1 1 CyDye DIGE Fluor minimal dyes CyDye DIGE Fluor minimal dyes consist of three bright spectrally resolvable fluors Cy2 Cy3 and Cy5 that are matched for mass and charge The fluors offer great sensitivity detecting as little as 125 pg of transferrin and giving a linear response to protein concentration of up to four orders of magnitude In comparison silver stain detects 1 60 ng of protein with less than a hundred fold dynamic range 90 91 Narrow excitation and emission bands mean that the CyDye Fluor minimal dyes are spectrally distinct which makes them ideal for multi color detection Fig 60 Most importantly the fluors are mass and charge matched so that the same protein labeled with any of the CyDye DIGE Fluor minimal dyes will migrate to the same position within a 2 D gel 92 94 The novel properties of the CyDye DIGE Fluor minimal dyes make them ideal for multiplexing different protein samples within the same 2 D gel This permits inclusion of an internal standard within each gel which limits experimental variation and ensures accurate intra and inter gel matching 1 488nm 900 BP4O Filters Cy2
160. ional reading and reference material Staining Tray Set provides a convenient means of staining up to four large format gels at a time film backed as well as unbacked The set includes two stainless steel trays and a perforated stainless steel tray which seats within the staining trays and a transparent plastic cover The perforated insert supports and restrains gels for transfer between staining trays while allowing staining solution to drain rapidly 106 80 6429 60 AD The film supported DALT and ExcelGel precast gels are optimally stored in sheet protectors after soaking them in 10 v v glycerol for 30 min Unbacked gels are shrunk back to their original sizes by soaking them in 30 v v methanol or ethanol 4 glycerol until they match their original sizes For autoradiography the gels are dried onto strong filter paper with a vacuum dryer 5 1 Blotting Second dimension gels can be blotted onto a nitrocellulose or polyvinylidene difluoride PVDF membrane for immunochemical detection of specific proteins or for chemical microsequencing GE Healthcare offers a range of blotting membranes and equipment for such purposes Hybond ECL is an unsupported 100 pure nitrocellulose membrane that has been validated for use with ECL Western Blotting System and for all radioactive non radioactive and chromogenic protein blotting applications It has excellent sensitivity resolution and low background Hybond P is a PVDF membrane optimized f
161. ions J Biochem Biophys Methods 6 317 339 1982 Gorg A et al The current state of two dimensional electrophoresis with immobilized pH gradients Electrophoresis 9 531 546 1988 Gorg A et al The current state of two dimensional electrophoresis with immobilized pH gradients Electrophoresis 21 1037 1053 2000 nl M et al Difference gel electrophoresis a single gel method for detecting changes in protein extracts Electrophoresis 18 2071 2077 1997 Wilkins M R et al From proteins to proteomes large scale protein identification by two dimensional electrophoresis and amino acid analysis Biotechnology 14 61 65 1996 Pennington S R et al Proteome analysis from protein characterization to biological function Trends Cell Bio 7 168 173 1997 Gorg A et al Two dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension IPG Dalt the state of the art and the controversy of vertical vs horizontal systems Electrophoresis 16 1079 1086 1995 Lenstra J A and Bloemendal H Topography of the total protein population from cultured cells upon fractionation by chemical extractions Eur J Biochem 135 413 423 1983 Molloy M P et al Extraction of membrane proteins by differential solubilization for separation using two dimensional gel electrophoresis Electrophoresis 19 837 844 1998 Ramsby M L et al Differential detergent fractionation of isolated he
162. ip gels must be rehydrated with the appropriate additives prior to IEF 3 IEE First dimension IEF is performed on a flatbed system at very high voltages with active temperature control 4 Immobiline DryStrip gel equilibration Strip equilibration in SDS containing buffer prepares the sample for the second dimension separation 5 SDS PAGE The strip is placed on the second dimension gel for SDS PAGE 6 Visualization Protein spots are stained to visualize them in the second dimension gel matrix Alternatively if the proteins were prelabeled the spots can be visualized by autoradiography by illumination of the gel with UV light or by using a fluorescence imager to detect the proteins 7 Analysis Analysis of the resultant two dimensional array of spots Equipment choices There are different options for methods and equipment for IEF and SDS PAGE Table 1 lists the instruments available from GE Healthcare For detailed information on the operation of any of the instruments described refer to the respective instrument user manual For other details about the instruments and related products refer to the GE Healthcare BioDirectory or visit www gehealthcare com Selecting an IEF system GE Healthcare offers two systems for first dimension separation Ettan IPGphor 3 Isoelectric Focusing System and Multiphor II Electrophoresis System Both are available with accessories for improving IEF performance The upgraded easy to use Ettan IPGph
163. is a neurotoxin and suspected carcinogen Read the manufacturer s safety data sheet MSDS detailing the properties and precautions for all chemicals in your laboratory These safety data sheets should be reviewed prior to starting the procedures described in this handbook General handling procedures for hazardous chemicals include using double latex gloves for all protocols Hazardous materials should be weighed in a fume hood while wearing a disposable dust mask Follow all local rules and regulations for handling and disposal of materials 80 6429 60 AD 15 16 80 6429 60 AD 1 Sample preparation 1 0 General strategy Appropriate sample preparation is absolutely essential for good 2 D electrophoresis results Due to the great diversity of protein sample types and origins the optimal sample preparation procedure for any given sample must be determined empirically Ideally the process will result in the complete solubilization disaggregation denaturation and reduction of the proteins in the sample There are several important differences in sample preparation for 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE See section 6 3 2 for more information When developing a sample preparation strategy it is important to have a clear idea of what is desired in the final 2 D result Is the goal to view as many proteins as possible or is only a subset of the proteins in the sample of potential interest Which is more important complet
164. is and other protein modifications Centrifuge and wash pellet with acetone or ethanol to remove residual TCA Proteins may be difficult to resolubilize and may not resolubilize completely Residual TCA must be removed by extensive washing with acetone or ethanol Extended exposure to this low PH solution may cause some protein degradation or modification Acetone precipitation This organic solvent is commonly used to precipitate proteins Many organic soluble contaminants e g detergents lipids will remain in solution Add at least three volumes of ice cold acetone to the extract Allow proteins to precipitate at 20 C for at least 2 h Pellet proteins by centrifugation 46 48 50 Residual acetone is removed by air drying or lyophilization Incomplete recovery of all proteins Compatibility of acetone with tubes may be an issue Precipitation with TCA in acetone The combination of TCA and acetone is commonly used to precipitate proteins during sample preparation for 2 D electrophoresis and is more effective than either TCA or acetone alone Suspend lysed or disrupted sample in 10 TCA in acetone with either 0 07 2 mercaptoethanol or 20 mM DTT Precipitate proteins for at least 45 min at 20 C Pellet proteins by centrifugation and wash pellet with cold acetone containing either 0 07 2 mercaptoethanol or 20 mM DTT Remove residual acetone by air drying or lyophilization 5 28 34 43 51 52
165. is of human serum is described in detail in section 1 5 3 24 80 6429 60 AD 1 4 Precipitation procedures Protein precipitation is an optional step in sample preparation for 2 D electrophoresis Precipitation followed by resuspension in sample solution is generally employed to selectively separate proteins in the sample from contaminating species such as salts detergents nucleic acids lipids etc that would otherwise interfere with the 2 D result Precipitation followed by resuspension can also be employed to prepare a concentrated protein sample from a dilute source e g plant tissues urine Note however that no precipitation technique is completely efficient and some proteins may not readily resuspend following precipitation Thus employing a precipitation step during sample preparation can alter the protein profile of a sample When complete and accurate representation of all the proteins in a sample is of paramount interest precipitation and resuspension should be avoided 2 D Clean Up Kit from GE Healthcare can be used to remove contaminating substances and improve the 2 D electrophoresis pattern Proteins are precipitated with a combination of precipitation reagents while the interfering substances such as nucleic acids salts lipids or detergents remain in solution Samples can be resuspended in the desired denaturing solution for IEF Each kit can process 50 samples of up to 100 ul each Section 1 4 1 describes the kit and pro
166. ithm of the molecular weight and the relative distance of migration of the SDS polypeptide complex Note This linear relationship is only valid for a certain molecular weight range which is determined by the polyacrylamide percentage See Table 24 for the optimum linear separation range for single percentage homogeneous and gradient gels 72 80 6429 60 AD The most commonly used buffer system for second dimension SDS PAGE is the Tris glycine system described by Laemmli 78 This buffer system separates proteins at high pH which confers the advantage of minimal protein aggregation and clean separation even at relatively heavy protein loads The Laemmli buffer system has the disadvantage of a limited gel shelflife Ettan DALT precast gels utilize a buffer system based on piperidinopropionamide PPA which combines long shelflife with the high separation pH of the Laemmli system Other buffer systems can also be used particularly the Tris tricine system of Schdgger and von Jagow 79 for improving resolution of polypeptides with M values below 10 000 ExcelGel precast gels for second dimension SDS PAGE on the Multiphor II Electrophoresis System see chapter 4 utilize a different Tris tricine buffer system 3 3 Electrophoresis using Ettan DALT Large Vertical electrophoresis systems Ettan DALTsix and DALTtwelve Large Vertical electrophoresis systems combined with 24 cm long Immobiline DryStrip gels offer the highest possible 2 D resolution B
167. its of this technique is presented in section 2 2 on page 45 e 2 D DIGE first described in 1997 by nl et al 5 offers a method for controlling system variations allowing biological variations and changes in protein expression to be identified with statistical confidence 80 6429 60 AD 7 e Automation of steps after 2 D electrophoresis such as gel image analysis spot picking spot digestion and target preparation for mass spectrometry have allowed a significant increase in the throughput of protein analysis and identification e New mass spectrometry techniques have been developed that allow rapid identification and characterization of very small quantities of peptides and proteins e More powerful less expensive computers and software are now available rendering thorough computerized evaluations of highly complex 2 D patterns to become economically feasible e Data about entire genomes of a number of organisms are now available allowing rapid identification of the gene encoding a protein separated by 2 D electrophoresis e Protein sequences are being added on a daily basis to databases available on the public domain Organizations such as the Human Proteome Organization HUPO are attempting to coordinate proteome analysis between many countries toward a common goal e The World Wide Web provides simple direct access to spot pattern databases for the comparison of electrophoresis results and genome sequence databases for a
168. k gels 1 2 0 45 lab cast and precast 2 17 max 100 4 00 1 5 mm thick gels 1 5 0 30 2 17 max 100 5 00 Ettan DALTtwelve set temperature to 25 C 1 mm thick gels 1 2 0 45 lab cast and precast 2 17 max 180 4 00 1 5 mm thick gels 1 5 0 30 2 17 max 180 6 00 Overnight runs in Ettan DALTsix set temperature to 30 C power supply for continuous run 1 0 mm thick gels run overnight 1 16 00 1 5 mm thick gels run overnight 15 18 30 Overnight runs in Ettan DALTtwelve set temperature to 30 C power supply for continuous run 1 0 mm thick gels run overnight tt 18 00 1 5 mm thick gels run overnight 15 17 00 For the best possible resolution faster separation times should be used Use the faster lt 6 h protocols instead 3 3 7 Preparing lab cast gels Wi Some of the chemicals used in the procedures that follow acrylamide N N methylenebisacrylamide ammonium persulfate TEMED thiourea DTT and iodoacetamide are very hazardous Acrylamide monomer for example is a neurotoxin and suspected carcinogen Read the manufacturer s safety data sheet MSDS detailing the properties and precautions for all chemicals in your laboratory These safety data sheets should be reviewed prior to starting the procedures described in this handbook General handling procedures for hazardous chemicals include using double latex gloves for all protocols Hazardous materials should be weighed in a fume hood while wearing a disposable dust mask Foll
169. kets Using this option step 4 can be reduced with the added kVh in step 1 to reach the specified total kVh t Follow steps 1 2 3a and 4a when using PGphor Regular Strip Holder or Cup Loading Strip Holder Follow steps 1 2 3b and 4b when using IPGphor Cup Loading Manifold 66 80 6429 60 AD Table 19 continued 24 cm strips Note When using IPGphor Manifold and 10 kV set current limit to 75 pA per strip and follow step 1 2 3b and 4b Using IPGphor Regular Strip Holder or Cup Loading Strip Holder with the 18 and 24 cm strips the maximum allowed voltage is 8000 V and current 50 pA per strip Follow step 1 2 3a 4a pH Step Voltage Voltage Time kVh intervals mode Vv h min 3 11 NL 1 Step and Hold 500 1 00 8 00 0 5 3 10 2 Gradient 1000 1 00 0 8 3a Gradientt 8000 3 00 13 5 4a Step and Holdt 8000 2 30 3 45 20 0 30 0 3b Gradient 10000 3 00 16 5 4b Step and Hold 10000 1 45 2 45 17 2 27 2 Total 35 0 45 0 3 10 NL 1 Step and Hold 500 1 00 7 00 0 5 3 7 NL 2 Gradient 1000 1 00 08 4 7 3a Gradientt 8000 3 00 13 5 3 5 6 NL 4a Step and Holdt 8000 3 45 5 36 30 0 45 0 3b Gradient 10000 3 00 16 5 4b Step and Hold 10000 2 45 4 15 27 2 42 2 Total 45 0 60 0 6 9 1 Step and Hold 500 1 00 5 00 0 5 7 11 NL 2 Gradient 1000 1 00 0 8 3a Gradientt 8000 3 00 13 5 4a Step and Holdt 8000 5 36 8 45 45 0 70 0 3b Gradient 10000 3 00 16 5 4b Step and Holdt 10000 4 15 6 45 42 2 67 2 Total 60 0 85 0 3 5 4 5 1 Ste
170. l 10 ammonium persulfate solution K 170 ul 170 ul 170 ul 170 ul 170 ul TEMEDt 17 ul 17 ul 17 ul 17 ul 17 ul Total volume 50 ml 50 ml 50 ml 50 ml 50 ml Heavy solution Final concentration 10 12 5 15 17 5 20 Monomer solution solution G 16 7m 21 0m 25 0 ml 29 2 ml 33 3 m 4x resolving gel buffer solution H 12 5 m 12 5 m 12 5 ml 12 5 ml 12 5 m Sucrose 7 59 7 59 7 59 7 59 7 59 10 SDS solution J 500 u 500 u 500 ul 500 ul 500 u Double distilled water 16 2m 11 7m 7 7 ml 3 5 ml om 10 ammonium persulfate solution K 165 u 165 u 165 ul 165 ul 165 u TEMED 16 5 u 16 5 u 16 5 ul 16 5 yl 16 5 u Total volume 50 ml 50 ml 50 ml 50 ml 50 ml Preparation of stock solutions is described in appendix I solutions G H J and K t Ammonium persulfate and TEMED are added immediately prior to casting the gel 3 4 3 Preparing miniVE SE 260 and SE 600 Ruby systems for electrophoresis For these electrophoresis units prepare enough 1x SDS electrophoresis buffer according to Table 32 preparation of stock solutions is described in appendix solution M Table 32 Gel tank buffer volumes for miniVE SE 260 and SE 600 Ruby electrophoresis systems System Gel tank buffer volume I miniVE 15 SE 260 0 6 SE 600 Ruby 5 Fill the anode buffer tank with SDS electrophoresis buffer Set the temperature if applicable 3 4 4 Equilibrating Immobiline DryStrip gels To equilibrate the Immobiline DryStrip gel see section 3 1 2 The equilibratio
171. l 17 1312 01 Glycerol 87 11 17 1325 01 SDS 100g 17 1313 01 Thiourea 100g RPN6301 odoacetamide 259 RPN6302 Tris 500g 17 1321 01 Urea 500 g 17 1319 01 CHAPS 1g 17 1314 01 Triton X 100 500 ml 17 1315 01 Dithiothreitol DTT 1g 17 1318 01 Bromophenol Blue 10g 17 1329 01 Bind Silane 25ml 17 1330 01 mmobiline DryStrip Cover Fluid 1l 17 1335 01 Amberlite IRN 150L 500g 17 1326 01 Enzymes Nuclease Mix 0 5 ml 80 6501 42 Deoxyribonuclease DNase 20mg 27 0516 01 Ribonuclease RNase A and RNase B 1g 27 0330 02 Ribonuclease A RNase A 100 mg 27 0323 01 80 6429 60 AD 155 Product Quantity Code No Molecular weight markers Peptide Marker Kit M range 2512 16 949 LMW SDS Marker Kit M range 14 400 97 000 HMW SDS Marker Kit M range 53 000 220 000 Full Range Rainbow Molecular Weight Markers pl calibration kit Carbamylyte Calibration Kit Automated gel and blot processing Processor Plus Base Unit includes Base Unit Reagent Tubing and Protocol Key Accessories to make functional for staining and or blotting Staining Tray Pack 19 x 29 cm complete with gel staining tray base tray and lid Staining Tray Pack 29 x 35 cm includes gel staining tray base tray and lid Blot Processing Tray Pack includes tray base disposable mini and standard trays lid reagent bottles and rack and vented lid for waste products Additional accessories for Processor Plus Stainless Steel Staining Tray 19 x 29
172. l and a buffer circulation pump provide a precise and uniform thermal environment Up to fourteen 1 mm thick gels can be cast simultaneously in the Ettan DALTtwelve Gel Caster For rapid results the mini gel units miniVE Fig 4 or SE 260 Fig 5 are recommended The second dimension separation is typically complete in 1 2 h The use of mini gels for the second dimension is ideal when quick profiling is required or when the protein pattern is relatively simple For increased throughput and resolution the standard sized SE 600 Ruby Vertical Electrophoresis System Fig 6 is recommended SE 600 Ruby accommodates up to four 16 cm long gels and the built in heat exchanger offers cooling capability in conjunction with MultiTemp IIl Thermostatic Circulator for increased reproducibility The standard spacer width is 2 cm giving a 14 cm wide gel If additional space for molecular weight markers is desired at both ends of a 13 cm Immobiline DryStrip gel 1 cm wide spacers are available for the preparation of 16 cm wide gels Short 8 cm clamps plates and spacers are available for preparing gels that are 14 16 cm wide and 8 cm long These short gels may be used for rapid simultaneous second dimensional analysis of many 7 cm Immobiline DryStrip gels Multiphor II Electrophoresis System The flatbed Multiphor Electrophoresis System Fig 7 provides excellent resolution and relatively rapid separations in a large format gel Precast ExcelGel products o
173. l group To avoid redox related streaking all the basic strips should be rehydrated with DeStreak Rehydration Solution see sections 2 6 1 and 2 6 2 Note The gradients overlap to enable the assembly of virtual high resolution 2 D maps from different narrow range separations If a specialized pH gradient is required recipes for preparing custom narrow and wide range immobilized pH gradients are given in references 72 and 73 Figure 19 shows typical results using broad and medium range 24 cm Immobiline DryStrip gels 80 6429 60 AD 45 Fig 19a Broad range Immobiline DryStrip pH 3 11 NL 24 cm using 0 5 IPG Buffer 3 11 NL run for 42 kVh Sample 100 ug mouse liver extract 7 5 ug alkylated lysozyme cup application at the anode Fig 19b Medium range Fig 19c Medium range Fig 19d Medium range Fig 19e Medium range Immobiline DryStrip Immobiline DryStrip Immobiline DryStrip Immobiline DryStrip pH 3 5 6 NL 24 cm using pH 5 3 6 5 24 cm using PH 6 2 7 5 24 cm using pH 7 11 NL 24 cm using 2 IPG Buffer 3 5 5 0 run 2 IPG Buffer 5 5 6 7 run 2 IPG Buffer 6 11 run 0 5 IPG Buffer 7 11 NL for 50 kVh for 116 kVh for 116 kVh run for 75 kVh Sample 140 ug mouse liver Sample 100 ug mouse liver Sample 100 ug mouse liver Sample 100 ug of mouse extract cup application at extract cup application at extract cup application at liver extract 5 ug alkylated the cathode the anode the anode lysozyme cup application at t
174. l pores Nucleic acids can bind to proteins through electrostatic interactions preventing focusing If the separated sample proteins are visualized by silver staining nucleic acids present in the gel will also stain resulting in a background smear on the 2 D gel Treat samples rich in nucleic acids with a protease free DNase RNase mixture to reduce he nucleic acids to mono and oligonucleotides This is often done by adding 0 1 times the volume of a solution containing 1 mg ml DNase 0 25 mg ml RNase A and 50 mM MgCl ollowed by incubation on ice 33 50 Note The DNase and RNase proteins may appear on the 2 D map Ultracentrifugation can be used to remove arge nucleic acids however this technique may also remove high molecular weight proteins from the sample When using low ionic strength extraction conditions negatively charged nucleic acids may form complexes with positively charged proteins High ionic strength extraction and or high pH extraction may minimize these interactions Note that salts added during extraction must be subsequently removed see above Polysaccharides Polysaccharides can clog gel pores causing either precipitation or extended focusing times resulting in horizontal streaking Some polysaccharides contain negative charges and can complex with proteins by electrostatic interactions Precipitate the sample in TCA ammonium sulfate or phenol ammonium acetate then centrifug
175. l sample application methods can be used but sample specific limitations may exist Use up to 10 mM reducing agent per 100 ul of sample With basic Immobiline DryStrip pH intervals 6 2 7 5 6 9 6 11 and 7 11 NL we recommend anodic cup application or anodic paper bridge loading Use up to 20 mM reducing agent per 100 ul of sample e Using rehydration loading on basic strips the sample in rehydration solution may contain up to 1 mM reducing agent This reducing power will be consumed during the rehydration step and early start of the run and thiols will be transferred to disulfides during the run 56 80 6429 60 AD Table 18 Rehydration solution volume per Immobiline DryStrip Ettan IPGphor 3 protocol Immobiline DryStrip Total volume per strip gel length cm ul 7 125 11 200 13 250 18 340 24 450 Including sample if applied Run Run the gels according to the instructions included with the Immobiline DryStrip package Protocol DeStreak Reagent Reagents supplied DeStreak Reagent 1 ml Required but not provided Rehydration solution without reducing agents sample buffer containing reducing agents up to 20 mM such as DTT 6 mercaptoethanol or tris 2 carboxyethyl phosphine TCEP 1 Prepare DeStreak Reagent for use by adding 12 pl 15 mg of DeStreak Reagent per ml of rehydration solution containing no reducing agents 2 Follow the steps for sample preparation application and gel run as
176. l separation of protein SAMPLES ssscsssssccceccccesssssssssssstnnnneeseseee 121 6 3 5 Summary of key differences between minimal labeling and saturation labeling 122 6 3 6 IMAJIN eaii a a aE eE a AiE E ET 122 6 3 7 Image analysis with DeCyder 2 D Differential Analysis Software sees 123 6 3 8 Further analysis Of Protein SPOS eecccccccsssssssscsecsssssssssssssnnsussssesseseececeeeeeesessssnsssssuusneseesseseeeees 123 6 4 Troubleshooting 2 D DIGE sesssssssssssssssssssscescecssesssssssssssnnssssesseseeeeeeeeeesessssssssssnsnussessesseseeeeeeeeessesssees 124 TATOUDICSHOOUIAG a A E A E spree 125 a a E O E ET A anterasseeancits ee 129 SOME OMS se a o E REE E A A AER 129 A Sample preparation solution with urea for 2 D electrophoresis esses 129 B Sample preparation solution with urea and thiourea for 2 D electrophoresis 129 C Urea rehydration Stock solution no ceccscsssscsssceccecssssssssssssunesesesseseeseeeessesssssssssnssusussseseseeeeeeeeeeees 130 D ThioUred rehydration StOCK SOLUCION sssini tinsi 130 E SDS equilibration buffer SOLUTION e ccceccccssssssssssssssssssessssssssssssecseeceeeesssssssssssumumssesseseeseeeeeeessessssssnsnniaee 130 F 10x Laemmli SDS electrophoresis DUFFEL ssssssssscccceccccecssssssssssumsssessesseseeeeeceseessssssnssnsees 131 G 30 T 2 6 C monomer stock SOLUTION on eeeeesesssssssssssssssseeceeeecesesssestsssttttenttstensnssensnsssssseeeeeeeeeeeeee 131 H 4x resolving g
177. l standard each labeled with a different dye on a 2 D gel AA Post stain gel 1st and 2nd e g Deep Purple dimension Total Protein Stain separation Preparative l DeCyder gel workflow l l Differential ifferentia Spotpiek Gel l eo i list processing i data l MALDI MS protein Analytical gel workflow identification Fig 64 Ettan DIGE system workflow 116 80 6429 60 AD 6 3 1 Experimental design for Ettan DIGE system applications The differences in methodology between 2 D DIGE and traditional 2 D electrophoresis are outlined in Table 41 Table 41 Differences in methodology between 2 D DIGE and traditional 2 D electrophoresis Step 2 D DIGE Traditional 2 D electrophoresis Sample preparation Exclude carrier ampholyte and Carrier ampholyte and reductant included reductant until after labeling Concentrate protein to 1 10 mg ml Concentration of sample is optional Labeling Addition of CyDye required No CyDye required Labeling reaction terminated No lysine addition required with addition of lysine 2x sample buffer added The sample buffer is made with Not required to sample after labeling 2x concentration of reductant and carrier ampholyte An equal volume of this buffer is added to the labeled proteins See the protocol in section 6 3 4 Electrophoresis Use low fluorescence glass plates Use standard glass plates Traditional 2 D electrophoresis suffers from
178. l to 850 ml water Washing solution may be prepared in advance and can be stored at room temperature for up to 6 months Note Ethanol 15 v v in water can be replaced by methanol 30 v v in water We recommend that you consistently use either methanol or ethanol throughout the procedure D Storage solution for gels Gels should be stored at 4 C protected from light in 1 citric acid Add 10 g citric acid to 1 water pH 2 3 For extended storage up to 6 months add Deep Purple 1 200 to the storage solution Protocol Low fluorescence glass plates should be used for plastic backed gels as these backed gels have problems with background Gel electrophoresis Perform electrophoresis according to established techniques Note If visual orientation is required on 1D gels Rainbow Markers RPN800 may be used If a tracking dye is used in the loading buffer such as bromophenol blue it is recommended to run the dye front just off the bottom of the gel 138 80 6429 60 AD Fixation 1 Place an appropriate volume of 15 ethanol 1 citric acid v v solution A into the containers that will be used to process gels The recommended volume of fixation solution required is 20 fold excess of the gel volume Table A Note Alternative fixation solutions that have been used successfully with Deep Purple Total Protein Stain are e 7 5 acetic acid 10 ethanol e 7 0 acetic acid 30 ethanol 2 Dismantle the electrophoresis apparatus
179. labeling dyes The minimal dyes are intended for general 2 D application use where sufficient amounts of sample are available The saturation dyes included in the scarce sample and preparative gel labeling kits are designed to be used for applications where only small amounts of sample are available for example in Laser Capture Microdissection Ettan DIGE system capitalizes on the ability to multiplex by combining CyDye DIGE Fluor dyes with DeCyder 2 D Differential Analysis Software DeCyder 2 D software has been designed specifically for Ettan DIGE applications It utilizes a proprietary co detection algorithm that permits automatic detection background subtraction quantitation normalization and inter gel matching of the fluorescent images The use of an internal standard gives an increased confidence that the results reflect true biological effects and are not due to system variation The system comprises CyDye DIGE Fluor dyes for protein labeling a choice of Ettan IPGphor 3 Isoelectric Focusing System or Multiphor II Electrophoresis System for first dimension separation Ettan DALTsix DALTtwelve or SE 600 Ruby vertical electrophoresis systems for second dimension separation Typhoon Variable Mode Imager for advanced imaging and DeCyder 2 D Differential Analysis Software for quantitation and statistical analysis of protein differences over a linear dynamic range of up to four orders of magnitude Figure 59 summarizes the steps in the analysis
180. lders and the Ettan IPGphor 3 unit which includes a high voltage DC power supply solid state temperature control using Peltier technology and programming options for up to 10 user defined IEF protocols IPG strips are available in five lengths 7 11 13 18 and 24 cm and a number of pH ranges both linear and nonlinear Section 2 2 discusses choices for length of strip pH gradient and buffer The Manifold accommodates IPG strips from 7 to 24 cm long and holds up to 12 strips It allows for three main means of sample application 1 rehydration loading generally for preparative or analytical loadings of broad range or narrow range strips 2 cup loading anodic or cathodic generally for analytical loadings of basic strips or very acidic strips respectively and 3 paper bridge loading anodic or cathodic generally for preparative loadings of basic strips or very acidic strips respectively Further discussion of these techniques can be found in section 2 7 The Manifold must be used in conjunction with the DryStrip IPGbox with or without sample included in order to rehydrate the Immobiline DryStrips see section 2 7 The Manifold can also be used for equilibrating the IPG strips prior to second dimension electrophoresis The regular Strip Holder see section 2 7 is placed on the Ettan IPGphor 3 electrode platform and the sample is introduced either during or after the rehydration step Up to 12 Strip Holders of the
181. le and place a Reswell Tray in the IPGbox Ensure that the Reswell Tray and the IPGbox Insert are clean and dry If required wipe the IPGbox Insert with a soft tissue moistened with 70 ethanol 3 Pipette the appropriate volume of selected rehydration solution and sample if applied evenly over the slots corresponding to the length of the DryStrip gels See Table 18 for rehydration solution volumes Strip lengths are measured from the straight ended part of the wells of the Reswell Tray and marked with lines on the top of the Reswell Tray Note For complete sample uptake do not apply excess rehydration solution 4 Carefully pull off the cover film from Immobiline DryStrip gel and place Immobiline DryStrip gel into the slot gel side down Distribute the rehydration solution evenly under the strip Gently lift and lower the strip and slide it back and forth along the surface of the solution to get complete and even wetting of the entire gel Note Ensure that no bubbles are trapped under the Immobiline DryStrip gel 60 80 6429 60 AD Fig 28 Example of IPG strips in a Reswell Tray 5 Gently close the lid of the IPGbox and allow the Immobiline DryStrip gels to rehydrate at room temperature for 10 24 hours overnight is recommended 6 Use the Immobiline DryStrip gels within 20 minutes of opening the IPGbox Discard the used Reswell Tray Note Do not store the Reswell Tray in IPGbox Long time pressure of a Reswell Tray can reduce the l
182. le preparation kits and reagents Sample Grinding Kit 50 samples 80 6483 37 Protease Inhibitor Mix 1ml 80 6501 23 illustra triplePrep Kit 50 preps 28 9425 44 Nuclease Mix 0 5 ml 80 6501 42 Albumin and IgG Removal Kit 10 samples RPN6300 2 D Clean Up Kit 50 samples 80 6484 51 2 D Quant Kit 500 assays 80 6483 56 2 D Protein Extraction Buffer Trial Kit 6x10ml 28 9435 22 Mini Dialysis Kit 1 kDa cut off up to 250 ul 50 samples 80 6483 75 Mini Dialysis Kit 1 kDa cut off up to 2 ml 50 samples 80 6483 94 Mini Dialysis Kit 8 kDa cut off up to 250 ul 50 samples 80 6484 13 Mini Dialysis Kit 8 kDa cut off up to 2 ml 50 samples 80 6484 32 Vivaspin 6 MWCO 50 000 25 pack 28 9323 18 Spectrophotometer Ultrospec 3100 pro UV Visible Spectrophotometer Inquire First dimension products and accessories Ettan IPGphor 3 Isoelectric Focusing System Ettan IPGphor 3 Isoelectric Focusing Unit 1 11 0033 64 Ettan IPGphor Manifold Complete 80 6498 38 IPGbox kit 1 28 9334 92 Sample cups 120 pk 80 6498 95 Paper electrode 240 pk 80 6499 14 Paper bridge pads 120 pk 80 6499 33 Multiphor II Electrophoresis System and accessories Multiphor II Electrophoresis Unit 18 1018 06 MultiTemp III Thermostatic Circulator 115 V 18 1102 77 MultiTemp III Thermostatic Circulator 230 V 18 1102 78 EPS 3501 XL Power Supply 18 1130 05 Immobiline DryStrip Kit and other accessories Immobiline DryStrip Kit for use with Multiphor II only 18 1004 30 Immobiline DryStrip Cover Fluid Jil 17 1335 01
183. linked to an acrylamide monomer The general structure of Immobiline reagents is CH3 CH C NH R II O R weakly acidic or basic buffering group Immobilized pH gradients are formed using two solutions one containing a relatively acidic mixture of acrylamido buffers and the other containing a relatively basic buffer mixture The concentrations of the various buffers in the two solutions define the range and shape of the pH gradient produced Both solutions contain acrylamide monomers and catalysts During polymerization the acrylamide portion of the buffers copolymerizes with the acrylamide and bisacrylamide monomers to form a polyacrylamide gel Figure 17 is a graphic representation of the polyacrylamide matrix with attached buffering groups 42 80 6429 60 AD Fig 17 Immobilized pH gradient polyacrylamide gel matrix showing attached buffering groups For improved performance and simplified handling the Immobiline DryStrip gel is cast onto a plastic backing GelBond PAGfilm The gel is then washed to remove catalysts and unpolymerized monomers that could otherwise modify proteins and interfere with separation Finally the gel is dried and cut into 3 mm wide strips The resulting Immobiline DryStrip gels can be rehydrated with a rehydration solution containing the necessary components for first dimension IEF IEF is performed with the Immobiline DryStrip gels using a flatbed electrophoresis unit such as Ettan IPGphor 3 The advanta
184. ml Heavy solution Final concentration 12 14 16 18 20 Monomer solution solution G 180 m 210m 240 ml 270 ml 300m 4x resolving gel buffer solution H 113m 113m 113 ml 113 ml 113m Glycerol 87 w w 31m 31m 31 ml 31 ml 31m 10 SDS solution J 45m 45m 45 ml 4 5 ml 45m Double distilled water 119 9m 89 9m 59 9 ml 29 9 ml om 10 ammonium persulfatet solution K 14m 14m 14 ml 14ml 14m TEMEDt 0 225m 0 225m 0 225 ml 0 225 ml 0 225m Total volume 450 ml 450 ml 450 ml 450 ml 450 ml Preparation of stock solutions is described in appendix solutions G H J and K Adjust as necessary for the thickness of the gels and the number of gels cast t Add after optional deaeration 82 80 6429 60 AD 3 3 8 Preparing Ettan DALT electrophoresis units for electrophoresis using lab cast gels For Ettan DALT electrophoresis units the lower tank requires 1x SDS electrophoresis buffer while the upper chamber requires buffer of a higher concentration Prepare the required buffers as described in Table 28 preparation of stock solutions is described in appendix solutions M and F v For Ettan DALT Gel 12 5 the DALT Buffer Kit must be used to prepare the anode and cathode buffers described in section 3 3 1 Table 28 Tank buffer solutions for Ettan DALT systems with lab cast Laemmli gels System Anodic buffer Volume Cathodic buffer Volume lower buffer chamber I upper buffer chamber ml Dilute from 10x stock Ettan DALTsix 1
185. mmediately or stored for up to three months at 70 C in the dark To confirm efficient labeling any new protein samples should be labeled with CyDye DIGE Fluor Cy5 minimal dye and run on a 1 D SDS PAGE gel to compare the efficiency of labeling against a control lysate already known to label successfully A lysate of known concentration in an Ettan DIGE system compatible lysis buffer would be a suitable alternative control 120 80 6429 60 AD 6 3 4 Two dimensional separation of protein samples Separation of labeled proteins is carried out using traditional 2 D polyacrylamide gel electrophoresis see chapters 2 4 Protocol A Combining protein samples for multiplexing Protein samples labeled with different CyDye DIGE Fluor minimal dyes are combined according to the experimental design see section 6 3 For best results one or two labeled protein samples usually Cy3 or Cy5 are combined with a labeled internal standard usually Cy2 which is a pool of aliquots of all biological samples in the experiment B Diluting labeled protein sample in sample buffer The sample mixture is diluted further in sample buffer prior to separating the individual proteins on a 2 D gel Ettan DIGE system compatible 2x sample buffer contains 7 M urea 2 M thiourea 2 CHAPS w v 2 IPG buffer or Pharmalyte v v for IEF 2 DTT w v Add 1 volume of 2x sample buffer to sample Mix and leave on ice for at least 10 min C Rehydrating Immobiline DryStrip
186. mobiline DryStrip IPGbox when the Manifold is used with Ettan IPGphor 3 and also when Multiphor II Electrophoresis System is used When using Ettan IPGphor 3 and standard Strip Holders the strips should be rehydrated in the Strip Holders themselves Rehydration solution which may or may not include the sample is applied to the reservoir channels of the Immobiline DryStrip IPGbox or Strip Holder and then the Immobiline DryStrip gels are soaked individually There are two general rehydration methods 1 passive rehydration in which no electric field is applied during the process and 2 active rehydration which is rehydration under low voltage 20 120 V Active rehydration can facilitate the entry into the strip of high molecular weight proteins 70 Passive rehydration can be done in the Strip Holder or IPGbox but active rehydration can be done only in the Strip Holder Procedures for using both Strip Holders and the IPGbox are described below in section 2 7 or The IPGbox a separate product is required for proper strip rehydration when using the Manifold The channel in the Manifold is too wide to ensure proper absorption of the required volumes of rehydration solution EF The lid of IPG box prevents the strips from drying during rehydration thus eliminating the possibility of urea crystallization and the need for Immobiline DryStrip Cover Fluid 80 6429 60 AD 53 2 6 1 Components of rehydration solution The choice of the most app
187. mple with a solution containing an excess of nonionic or zwitterionic detergent OR e Separate the SDS from the sample protein by acetone precipitation Enzymatic lysis 23 24 Cells with cell walls can be lysed gently Plant tissue Treat cells with enzyme in an iso osmotic solution following enzymatic removal of the cell wall bacterial cells This must be done with an enzyme specific fungal cells for the type of cell to be lysed e g lysozyme for bacterial cells cellulase and pectinase for plant cells lyticase for yeast cells 20 80 6429 60 AD 1 1 2 More vigorous lysis methods These methods are employed when cells are less easily disrupted i e cells in solid tissues or cells with tough cell walls More vigorous lysis methods will result in complete disruption of the cells but care must be taken to avoid heating or foaming during these procedures Table 6 summarizes these options Table 6 More vigorous lysis methods Cell disruption method Application General procedure Sonication 5 25 26 Ultrasonic waves generated by a sonicator Cell suspensions Sonicate cell suspension in short bursts to lyse cells through shear forces avoid heating Complete shearing is obtained when Cool on ice between bursts maximal agitation is achieved but care must be taken to minimize heating and foaming French pressure cell 23 24 27 Cells are lysed by shear forces resulting Microorganisms Place cell suspension
188. mplete proceed to the second dimension separation immediately or store the Immobiline DryStrip gels at 60 C or below This can be conveniently done by placing the strips between plastic sheets as suggested by Gorg et al 3 or on glass plates covered in plastic wrap Alternatively the DryStrip gels can be stored in screw cap tubes The 7 cm strips fit in disposable 15 ml conical tubes 11 13 and 18 cm strips fit in 25 x 200 mm screw cap culture tubes and 18 and 24 cm strips fit in Equilibration Tubes see ordering information The equilibration process is discussed in section 3 1 80 6429 60 AD 99 4 1 9 Troubleshooting A Table 38 lists possible problems that could be encountered during IEF using Multiphor II Electrophoresis System and how to solve them Table 38 Troubleshooting first dimension IEF Multiphor II Electrophoresis System and Immobiline DryStrip Kit Symptom Possible cause Remedy Sample cups leak Incorrect handling and placement of sample cups Sample cups are fragile and should not be used too many times Make sure the sample cups are aligned with the Immobiline DryStrip gels Make sure the bottoms of the sample cups are flat against the surface of the Immobiline DryStrip gels Note Leaks can often be detected prior to sample application Observe the Immobiline DryStrip Cover Fluid when it is poured into the Immobiline DryStrip Kit tray If it leaks in through the bottom of the sample cup
189. n J X et al Postelectrophoretic staining of proteins separated by two dimensional gel electrophoresis using SYPRO dyes Electrophoresis 21 3657 3665 2000 Lopez M F et al A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two dimensional gels and identification by peptide mass profiling Electrophoresis 21 3673 3683 2000 Tonge R et al Validation and development of fluorescence two dimensional differential gel electrophoresis proteomics technology Proteomics 1 377 396 2001 Ruepp S U et al Genomics and proteomics analysis of acetaminophen toxicity in mouse liver Toxicol Sci 65 135 150 2002 Zhou G et al 2D differential in gel electrophoresis for the identification of esophageal scans cell cancer specific protein markers Mol Cell Proteomics 1 117 123 2002 Alban A et al A novel experimental design for comparative two dimensional gel analysis two dimensional difference gel electrophoresis incorporating an internal standard Proteomics 3 36 44 2003 Yan J et al Fluorescence 2 D difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli Proteomics 2 1682 1698 2002 Syrovy and Hodny Z Staining and quantification of proteins separated by polyacrylamide gel electrophoresis J Chromat 569 175 196 1991 Gharbi S et al Evaluation of two dimensional differential gel electrophoresis for proteomic expressi
190. n IPGphor 3 platform is available in three application based options for high throughput analytical and micropreparative protein analysis Table 15 summarizes these options Table 15 Several options based on application The complete solution Ettan IPGphor 3 Isoelectric Focusing Unit plus Ettan IPGphor Manifold IPGbox ceramic Strip Holders to run 7 11 13 18 or 24 cm IPG strips and appropriate IPG strips and buffers For high throughput micropreparative applications Ettan IPGphor 3 Isoelectric Focusing Unit plus Ettan IPGphor Manifold IPGbox and appropriate IPG strips and buffers For analytical study of protein profiles Ettan IPGphor 3 Isoelectric Focusing Unit plus ceramic Strip Holders and appropriate IPG strips and buffers 80 6429 60 AD 49 E Fig 23 Summary of the steps involved in first dimension IEF using Ettan IPGphor 3 IPGbox and Manifold 50 80 6429 60 AD 1 Rehydrate IPG strips Rehydrate Immobiline DryStrip gels with the gel side down in the appropriate volume of rehydration solution using IPGbox Allow the IPG strips to rehydrate overnight 10 20 h 2 Position Manifold Position the Manifold on Ettan IPGphor 3 The small T shaped protrusion fits into the cutout section of the Ettan IPGphor 3 bed making positioning easy 3 Transfer IPG strips to Manifold Pour the appropriate volume 108 ml of Immobiline DryStrip Cover Fluid evenly in all the channels Transfer the IPG st
191. n an immense dynamic range of relative abundance and a variety of physicochemical properties including solubility hydrophobicity hydrophilicity size and or charge Consequently sample preparation and prefractionation are actually in the focus of interest combined with new instrumentation for multiple runs and high throughput analysis Is there perfection in view There are still some challenges in the state of the art technology of 2 D electrophoresis but less than often expected and repeatedly described It is my pleasure to introduce the third edition of a most successful manual on 2 D electrophoresis It clearly describes the actual techniques for 2 D electrophoresis with IPG strips which should be stringently controlled and provides detailed protocols for newcomers as well as for experienced users New techniques such as 2 D DIGE and different sample preparation methods are included Finally there is a most valuable comprehensive pictorial troubleshooting guide just in case Murphy s Law something went wrong Angelika Gorg Technical University of Munich Germany September 2004 Contents PROPO CO ETEA EEA EEA EEE E A TET 2 TFODUCEIOM nAaR E TEEN 7 introd ctonto this handBoOK kisenes ennn n A E iN 7 Introdu ti nito 2 D electrophoreSiS unisini inis 7 Symbols used in this NANGDOOK cesesssessssssssssscseccccccccescssssssnssnssnsunsssseseeceeseeeeceeeessessssssssssnnusstsesseseeeeeeeeeeeese
192. n procedure is the same whether applying the strip to precast or lab cast gels 86 80 6429 60 AD 3 4 5 Applying Immobiline DryStrip gels Protocol 1 Dip the Immobiline DryStrip gel in SDS Buffer 2 While the SDS gels still are in the gel caster apply the Immobiline DryStrip gels on top of them Push the strips gently down to the gel surface 3 Seal the Immobiline DryStrip gel in place with melted agarose See appendix solution N Agarose sealing solution 3 4 6 Inserting gels into miniVE SE 260 and SE 600 Ruby systems Protocol miniVE For detailed information refer to the miniVE user manual 1 Make sure the sealing plate is in the half open position 2 Lower each module into the tank seating it in the locating slots 3 Add the appropriate amount of electrophoresis buffer to the tank and to the upper buffer chamber 4 Attach or close the lid and connect the power leads to the power supply Protocol SE 260 system For detailed information refer to the SE 260 user manual 1 Apply the SDS gel to the electrophoresis tank 2 Clamp the gel in position and fill up the anode and cathode buffer chambers 3 Attach or close the lid and connect the power leads to the power supply Protocol SE 600 Ruby system For detailed information refer to the SE 600 Ruby user manual 1 Fit the slotted gasket in the upper buffer chamber 2 Put the upper buffer chamber onto the gel sandwiches in the casting stand Fix the gel
193. n the Immobiline Fig 49 Alignment of electrodes over Immobiline DryStrip gels DryStrip aligner 4 1 3 Sample application by cup loading EF If the sample was not applied by means of the rehydration solution it can be applied using the sample cups immediately prior to isoelectric focusing When sample cups are used the sample load limits are lower and more specific Guidelines on suitable sample loads for different gradients and Immobiline DryStrip gels are given in Table 16 see section 2 5 These values should only be regarded as a rough guide Suitable sample loads will vary greatly between samples and with the sensitivity of the staining method used Protocol 1 Prepare the sample Prepare the sample in a solution similar in composition to the rehydration solution used 2 Determine the point of sample application The optimal application point depends on the characteristics of the sample When the proteins of interest have acidic pls or when SDS has been used in sample preparation sample application near the cathode is recommended Anodic sample application is necessary with pH 6 11 and 6 9 gradients and preferred when pH 3 10 gradients are used The optimal application point can vary with the nature of the sample Empirical determination of the optimal application point is best 3 Position the sample cup bar Place sample cups on the sample cup bar high enough on the bar to avoid touching the gel surface Position the sample cup bar s
194. ng IPGphor 3 Control Software and uploaded to the instrument instantly Important safety features ensure safe high voltage runs Key accessories include Strip Holders IPGbox and Manifold Ettan IPGphor 3 provides e Integral 10 000 V power supply e Peltier solid state temperature control 18 25 C e Accommodation of one Manifold or 1 12 Strip Holders for 7 11 13 18 or 24 cm IPG strips e Programmable controller for voltage current temperature and time Fig 21 Ettan IPGphor 3 80 6429 60 AD 47 2 3 1 Ettan IPGphor 3 Control Software Ettan IPGphor 3 Control Software Fig 22 with an external personal computer Windows connected via a serial port can be used to control up to four Ettan IPGphor 3 units simultaneously each running a different set of run parameters With the software it is possible to e Create save and edit protocols Monitor voltage current and volt hours of the run and generate graphical display as the run proceeds e Open and view stored log files of previous runs e Start stop and pause Ettan IPGphor 3 e Generate status report on Ettan IPGphor 3 instantaneous run condition report on request e Enable Web browser remote monitoring of IPGphor 3 e Export log files to programs such as Microsoft Excel e Create professional reports that can be saved printed and exported Instrument log Aaetthas Bicectencer Ertan IJOphor SL mepmribsid sov Cemdtanm 60000 3 Step n hold 0000 O LIV
195. nimal dyes and Cy3 and Cy5 saturation dyes that have been designed to be both mass and charge matched As a consequence identical proteins labeled with each of the CyDye DIGE Fluor dyes will migrate to the same position on a 2 D gel This ability to separate more than one sample on a single gel permits the inclusion of up to two samples and an internal standard internal reference in every gel The internal standard is prepared by mixing together equal amounts of each sample in the experiment and including this mixture on each gel CyDye DIGE Fluors are e Size and charge matched The same labeled protein from different samples will migrate to the same position regardless of the dye used e pH insensitive No change in signal over the wide pH range used during first dimension separation IEF and equivalent migration in SDS gels e Spectrally resolvable The distinct signal from each fluor contributes to the accuracy e Highly sensitive and bright e Photostable There is minimal loss of signal during labeling separation and scanning Protein standard Protein extract 1 Protein extract 2 Label with Cy2 Label with Cy3 Label with Cy5 Mix labeled extracts I Protein separation Image analysis Overlay images Image analysis Data quantitation Fig 58 Multiplexing using the CyDye DIGE Fluor minimal dye option with Ettan DIGE system 80 6429 60 AD 111 CyDye DIGE Fluors are available as minimal and saturation
196. nsion disturbances Bases or buffers should be diluted to 5 mM or lower prior to loading the sample onto first dimension IEF 1 6 2 Examples of sample preparation solutions A widely used sample solution which can be used for initial experiments with an unknown sample is described in appendix solution A To solubilize more hydrophobic proteins use solution B in appendix For a general review of protein solubilization for electrophoretic analysis see reference 9 1 7 Quantitating protein samples Electrophoresis of protein samples requires accurate quantitation of the sample to be analyzed to ensure that an appropriate amount of protein is loaded In addition accurate quantitation facilitates comparison between similar samples by allowing identical amounts of protein to be loaded Accurate quantitation of samples prepared for electrophoresis is however difficult because many of the reagents used to prepare and solubilize samples for electrophoresis including detergents reductants chaotropes and carrier ampholytes are incompatible with common protein assays Current spectrophotometric methods for protein quantitation rely either on Coomassie brilliant blue binding 65 or protein catalyzed reduction of cupric Cu ion to cuprous Cu ion 66 68 Dye binding assays cannot be used in the presence of any reagent that also binds Coomassie brilliant blue This includes carrier ampholytes such as Pharmalyte and IPG Buffer and detergents su
197. nsure cassette s are properly closed properly closed 80 6429 60 AD 89 90 80 6429 60 AD 4 Use of the flatbed Multiphor II Electro phoresis System for first and second di mensions 4 0 Overview Multiphor II Electrophoresis System is a versatile flatbed system that provides excellent resolution and rapid separations in large format gels that are efficiently and uniformly cooled through a ceramic cooling plate connected to the cooling unit This improves resolution and speed at high voltages The modular design of the Multiphor II Electrophoresis System gives it the flexibility to handle virtually any flatbed electrophoretic technique It is particularly well suited for ultra thin gels 0 1 0 5 mm on glass or plastic supports up to sizes of 20 x 26cm Multiphor II Electrophoresis System comprises a buffer tank with four leveling feet ceramic aluminum oxide cooling plate with accessories polycarbonate safety lid and electrode holder with movable EPH IEF electrodes for buffer strips and electrode strips In addition to accommodating gels of different sizes the electrodes make secure uniform contact with buffer strips eliminating the need for large volumes of liquid buffers Buffer strips can be positioned and held in place using Multiphor II Buffer Strip Positioner To complete the Multiphor II Electrophoresis System EPS 3501 XL Power Supply and MultiTemp III Thermostatic Circulator are also required As described b
198. o achieve a concentration of 1 nmol ul see specification sheet supplied with the fluor e g add 25 pl DMF to 25 nmol of fluor Mix vigorously and centrifuge to collect fluor at the bottom of the vial The concentrated stock solution is stable for two months at 20 C or until the expiry date if sooner 2 Dilute CyDye DIGE Fluor minimal dyes to a working stock concentration Dilute the concentrated stock solutions to a working fluor concentration of 400 pmol ul using DMF e g add 2 ul of concentrated stock fluor to 3 ul of DMF The working fluor solution is stable for one week at 20 C or until the expiry date if sooner B Labeling protein sample with CyDye DIGE Fluor minimal dyes A pooled internal standard should be created from all of the samples This will need to be sufficient for inclusion on every gel It is recommended that the ratio of protein to fluor is maintained at 50 ug protein to 400 pmol fluor However it may be necessary to determine the optimum ratio for individual samples 1 Label proteins Add 1 ul of working fluor solution 400 pmol l to a volume of sample containing 50 yg of protein Mix thoroughly by vortexing Centrifuge to collect labeling mixture at the bottom of the tube Incubate on ice for 30 min in the dark 2 Quench the labeling reaction Add 1 ul of 10 mM lysine to stop the labeling reaction Mix well and leave on ice for 10 min in the dark 3 Store sample The labeled sample can either be processed i
199. o four gels glass plate size 18 x 16 cm Spacer 1 0 mm 1 cm wide 2 pk 80 6179 94 Spacer 1 0 mm 2 cm wide 2 pk 80 6180 70 Spacer 1 5 mm 1 cm wide 2 pk 80 6180 13 Spacer 1 5 mm 2 cm wide 2 pk 80 6180 89 SE 615 Multiple Gel Caster for 2 to 10 gels glass plate size 18 x 16 cm 80 6182 79 Glass plates 18 x 8 cm 2 pk 80 6186 59 Glass plates 18 x 8 cm low fluorescence 2 pk 80 6475 77 Divider glass plate 18 x 8 cm notched 80 6186 78 Glass plates 18 x 16 cm 2 pk 80 6178 99 Glass plates 18 x 16 cm low fluorescence 2 pk 80 6442 14 Divider glass plate 18 x 16 cm notched 80 6179 18 Clamp assembly 8 cm 2 pk 80 6187 35 Clamp assembly 16 cm 2 pk 80 6173 29 80 6429 60 AD 153 154 80 6429 60 AD Product Quantity Code No Ettan DALT Large Vertical Systems and accessories Ettan DALTtwelve Separation Unit and 80 6466 46 Power Supply Control Unit 115 VAC Ettan DALTtwelve Separation Unit and 80 6466 27 Power Supply Control Unit 230 VAC Ettan DALTsix Separation Unit and 80 6485 08 Power Supply Control Unit 115 VAC Ettan DALTsix Separation Unit and 80 6485 27 Power Supply Control Unit 230 VAC Ettan DALTtwelve Gel Caster complete includes 5 filler and 80 6467 22 16 separator sheets order cassettes separately Ettan DALTsix Gel Caster complete includes 6 filler and 80 6485 46 7 separator sheets order cassettes separately Ettan DALT Cassette Removal Too 2 pk 80 6474 82 Ettan DALT Buffer Seal Removal Tool
200. o the sample cups are a few millimeters away from the cathodic or anodic electrode depending on your sample The sample cups must face the electrode The sample cup bar has a spacer on one side slide the sample cup bar toward the anode cathode until the spacer just touches the anodic cathodic electrode 4 Press the sample cups against the Immobiline DryStrip gels Move the sample cups into position one sample cup above each Immobiline DryStrip gel and gently press the sample cups down to ensure good contact with each Immobiline DryStrip gel Fig 50 This is the most critical part of the setup Check that strips are in their correct straight position in the Immobiline DryStrip aligner 94 80 6429 60 AD 5 Apply Immobiline DryStrip Cover Fluid Once the sample cups are properly positioned pour 70 80 ml Immobiline DryStrip Cover Fluid into the tray to completely cover the Immobiline DryStrip gels If the Immobiline DryStrip Cover Fluid leaks into the sample cups remove it with a pipette correct the leakage and check for leakage again Add approximately 150 ml of Immobiline DryStrip Cover Fluid to completely cover the sample cups The Immobiline DryStrip gels are submerged under a layer of Immobiline DryStrip Cover Fluid to prevent drying of the gel precipitation of the components of the rehydration solution and diffusion of gas into the gel 6 Apply the sample Apply sample up to 100 ul per Immobiline DryStrip gel into the sample cups by pipet
201. ods Preface Despite alternative technologies that have emerged 2 dimensional 2 D electrophoresis is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures Furthermore it delivers a map of intact proteins that reflects changes in protein expression level isoforms or post translational modifications Last but not least today s 2 D electrophoresis technology with immobilized pH gradients IPGs has overcome the former limitations of carrier ampholyte based 2 D electrophoresis with respect to reproducibility handling resolution and separation of very acidic and or basic proteins NEPHGE The development of IPGs up to pH 12 together with an optimized protocol has enabled the analysis of very alkaline proteins and the construction of the corresponding databases Narrow overlapping IPGs provide increased resolution Ap 0 001 and in combination with prefractionation methods the potential for the detection of low abundance proteins The technique of 2 D electrophoresis with IPG strips has been constantly refined It is now readily available to many laboratories and is more or less routine Moreover Difference Gel Electrophoresis DIGE has proved to be a most powerful and exciting technique for the reliable detection and quantitation of differentially expressed proteins However there are still challenges with respect to proteomic samples that spa
202. oes not depend on reaction with protein side groups reactivity is largely independent of amino acid composition There is little protein to protein variation using this assay 38 80 6429 60 AD 1 7 1 Protein determination using 2 D Quant Kit 2 D Quant Kit is designed to accurately determine protein concentrations in samples for electrophoresis Proteins are quantitatively precipitated while interfering substances are left in solution The color density that develops in the 2 D Quant Kit procedure is inversely related to the protein concentration with a linear response to protein in the range of 0 50 ug and a volume range of 1 50 ul The procedure is compatible with common sample preparation reagents listed in Table 13 Table 13 Compounds tested for assay compatibility Compound Concentration SDS 2 w v CHAPS 4 w v Triton X 100 1 w v Pharmalyte pH 3 10 2 v v PG Buffer pH 3 10 NL 2 v v Tris 50 mM EDTA 10 mM DTT 1 65 mM 2 mercaptoethanol 2 v v Urea 8M Thiourea 2M Glycerol 30 w v 0 84 Standard curve with BSA dissolved in water J 4 Standard curve with BSA dissolved in first dimension sample solution 8 M urea 4 CHAPS 40 mM DTT 0 74 2 Pharmalyte pH 3 10 S064 0 0 54 0 44 0 10 20 30 40 50 ug BSA Fig 15 The 2 D Quant Kit protein assay eliminates interference from sample solution components Protocol 2 D Quant Kit Components supplied Precipitan
203. of the strip is toward the front edge of the gel 3 Position sample application pieces Using forceps place one IEF sample application piece at the end of each Immobiline DryStrip gel underneath the plastic tab formed by the overhanging gel support film at each end of the Immobiline DryStrip gel Be sure the application pieces touch the ends of the Immobiline DryStrip gel Fig 55 Note Application pieces absorb water that flows out of the Immobiline DryStrip gels during electrophoresis 4 Ensure contact between Immobiline DryStrip gel and ExcelGel Make sure that the Immobiline DryStrip gel is in full direct contact with the SDS gel To remove any bubbles stroke the plastic backing of the Immobiline DryStrip gel gently with a spatula or forceps 102 80 6429 60 AD 5 Optional Apply molecular weight marker proteins If loading marker proteins place an extra application piece on the surface of the gel just beyond the end of the Immobiline DryStrip gel Pipette the markers onto the extra sample application piece Apply the markers in a volume of 15 20 ul For less volume cut the sample application piece proportionally The markers should contain 200 1000 ng of each component for Coomassie staining and approximately 10 50 ng of each component for silver staining 6 Position electrodes Place the IEF electrode holder on the electrophoresis unit in the upper position and align the electrodes with the center of the buffer strips Plug in th
204. oftware ImageQuant TL mageQuant TL single user license 1 28 9236 62 mageQuant TL 5 user network license 1 28 9206 39 mageQuant TL 10 user network license 1 28 9236 57 mageQuant TL 7 0 and 1 28 9380 94 mageQuant TL SecurlTy 8 0 Software Package with Getting Started Guide mageQuant TL 7 0 and 1 28 9332 73 mageQuant TL SecurlTy 8 0 single user license ImageMaster 2D Platinum mageMaster 2D Platinum 7 0 DIGE 1 license 1 28 9380 55 mageMaster 2D Platinum 7 0 upgrade to DIGE 1 license 1 28 9398 10 mageMaster 2D Platinum 7 0 1 license 1 28 9380 91 mageMaster 2D Platinum 6 0 upgrade to 7 0 1 license 1 28 9399 70 mageMaster 2D Platinum 7 0 software package 1 28 9408 30 DeCyder 2D DeCyder 2D Oracle 10gR2 5 user license 1 28 9435 88 DeCyder 2D 7 0 1 user license 1 28 9442 75 DeCyder 2D 7 0 additional 1 user license 1 28 9442 77 DeCyder 2D 7 0 1 user trial license 1 28 9442 79 DeCyder 2D 7 0 1 user license upgrade from 6 5 2D 1 28 9442 80 DeCyder 2D 7 0 1 user license upgrade from 6 5 EDA 1 28 9442 81 DeCyder 2D 7 0 1 28 9435 86 pre installed computer 1 user license computer with DeCyder pre installed Software Package and e license 1 user node locked DeCyder 2D 7 0 Software Package 1 28 9435 83 Installation Guide Getting Started Guide and DeCyder 2D DVD case with installation disc packaged in DeCyder box DeCyder 2D 7 1 SPN 1 user license 1 29 9763 18 DeCyder 2D 7 1 SPN 1 user trial license 1 28 9763 20 DeCyde
205. om temperature Do not over dry cae Over dried pellets can be difficult to resuspend 7 Resuspend precipitated samples in an appropriate buffer as required Note Typical levels of protein recovered from a 15 pl serum sample are 150 220 ug 8 If samples are not to be used immediately store at 20 C or 70 C until required 1 6 Composition of sample preparation solution In order to achieve a well focused first dimension separation sample proteins must be completely disaggregated and fully solubilized Regardless of whether the sample is a relatively crude lysate or additional sample precipitation steps have been employed the sample solution must contain certain components to ensure complete solubilization and denaturation prior to first dimension IEF These always include urea and one or more detergents Complete denaturation ensures that each protein is present in only one configuration and that aggregation and inter molecular interaction is avoided 1 6 1 Components of sample preparation solutions The role of each component of the sample solution is described below as well as the recommended concentration range Denaturant IEF performed under denaturing conditions gives the highest resolution and the cleanest results Urea a neutral chaotrope is used as the denaturant in the first dimension of 2 D electrophoresis and is always included in the 2 D sample solution at a concentration of at least 8 M Urea solubilizes and unfolds
206. on second dimension gel Ensure that the Immobiline DryStrip gel is placed gel side down plastic backing upward on the SDS second dimension gel Detection method was not sensitive enough Use another detection method e g silver staining instead of Coomassie blue staining Failure of detection reagents Prepare fresh staining solutions Protein carbamylation Check expiry dates on staining solutions Do not heat any solutions containing urea above 30 C as cyanate a urea degradation product will carbamylate proteins changing heir pl Protein oxidation Use DeStreak Rehydration Solution During equilibration add DTT in first step to reduce the disulfide Add iodoacetamide in he second step to alkylate the thiol groups o prevent proteins from reoxidizing continues on following page 80 6429 60 AD 125 Table 44 Troubleshooting 2 D results continued Symptom Possible cause Remedy Distortion of 2 D pattern Vertical gel format The top Immediately after pouring the gel overlay surface of the second the surface with water saturated 1 butanol dimension gel is not flat Vertical gel format Uneven Degas the gel solution polymerization of gel due to incomplete polymerization Polymerization can be accelerated by too rapid polymerization increasing by 50 the amount of ammonium or leakage during gel casting persulfate and TEMED used Polymerization can be slowed by
207. on analysis of a model breast cancer cell system Mol Cell Proteomics 1 91 98 2002 148 80 6429 60 AD Additional reading and reference material Item literature type in parentheses Code No 2 D electrophoresis a comparison of carrier ampholyte 80 6419 53 and immobilized pH gradients scientific poster Automated staining of polyacrylamide gels with Hoefer Processor Plus 80 6343 34 technical manual Blot processing with the Processor Plus handbook 80 6447 27 Comparison of Deep Purple Total Protein Stain and Sypro Ruby in 1 D 18 1177 44 and 2 D gel electrophoresis application note CyDye DIGE Fluors and Labeling Kits data file 18 1164 84 DeCyder 2 D Differential Analysis Software v 7 0 data file 28 4015 43 DIGE Gel and DIGE Buffer Kit data file 28 9480 26 Detection and mass spectrometry identification of protein changes in 18 1177 51 low abundance tissue using CyDye DIGE Fluor saturation dyes data file Electrophoresis in Practice 18 1124 59 Westermeier R Wiley VCH Verlag GmbH Weinheim 2001 DALTsix Large Vertical Electrophoresis System for second 80 6490 02 dimension 2 D electrophoresis data file Fluorescence Imaging principles and methods handbook 63 0035 28 mageMaster 2 D Platinum data file 18 1177 20 mmobiline DryStrip Gels data file 18 1177 60 mmobiline DryStrip visualization of pH gradients technical brochure 18 1140 60 mmobiline DryStrip instructions 28 9537 55 mpro
208. on just prior to use t If necessary the concentration of urea can be increased to 9 M or 9 8 M Other neutral or zwitterionic detergents may be used at concentrations up to 2 w v Examples include Triton X 100 NP 40 octyl glucoside and the alkylamidosulfobetaine detergents ASB 14 and ASB 16 Calbiochem 5 Asan alternative to IPG Buffer use Pharmalyte 3 10 for Immobiline DryStrip 3 10 or 3 10 NL Pharmalyte 5 8 for Immobiline DryStrip 4 7 TA Pharmalyte IPG Buffer concentration of 0 5 125 ul is recommended with Ettan IPGphor 3 Isoelectric Focusing System and an IPG Buffer Pharmalyte concentration of 2 500 ul is recommended with the Multiphor II and Immobiline DryStrip Kit system Store in 2 5 ml aliquots at 20 C D Thiourea rehydration stock solution 7 M urea 2 M thiourea 2 CHAPS 0 5 2 Pharmalyte or IPG Buffer 0 002 bromophenol blue 25 ml Final concentration Amount Urea FW 60 06 7M 10 59 Thiourea FW 76 12 2M 3 8g CHAPSt 2 w v 059g Pharmalyte or IPG Buffer 0 5 v v or 2 v v 125 ul or 500 ul 1 Bromophenol blue stock solution 0 002 50 ul Double distilled water to 25 ml 13 5 ml required DTT is added just prior to use Add 7 mg DTT per 2 5 ml aliquot of rehydration stock solution t Other neutral or zwitterionic detergents may be used at concentrations up to 2 w v Examples include Triton X 100 NP 40 octyl glucoside and the alkylamidosulfobetaine detergents
209. on may be added to the tube following grinding up to 1 ml 5 Separate resin and debris by centrifugation for 5 10 min at maximum speed Fig 8C 6 Collect the supernatant and transfer to another tube Fig 8D If desired proceed with further clean up steps using 2 D Clean Up Kit section 1 4 1 1 1 4 Preparing samples from difficult protein sources To prepare proteins from tissues that are dilute sources of protein and contain high levels of interfering substances e g plant tissues the following procedure is recommended This method produces protein solutions substantially free of salts nucleic acids and other contaminants 1 Grind tissue in mortar and pestle with liquid nitrogen 2 Suspend powder in 10 TCA with 0 3 DTT in acetone 3 Keep at 18 C overnight and centrifuge Wash pellet with acetone 4 Dry and resuspend in 9 M urea 2 CHAPS 1 DTT 2 Pharmalyte 3 10 52 64 Samples should remain in sample solution at room temperature for at least 30 min for full denaturation and solubilization prior to centrifugation and subsequent sample application Heating of the sample in the presence of detergent can aid solubilization but should only be done prior to the addition of urea Sonication helps speed up solubilization particularly from material that is otherwise difficult to resuspend 22 80 6429 60 AD 1 2 Protecting against proteolysis When cells are lysed proteases are often liberated or activated Degrad
210. on procedures and improve sample quality which is essential for obtaining good electrophoresis results Table 4 summarizes the kits available these kits are described in more detail in following sections of this chapter Table 4 Sample Preparation Kits Product Quantity Use Sample Grinding Kit 50 samples disrupts up to 100 mg tissue or cell sample Protease Inhibitor Mix 1ml inhibits proteases 2 D Clean Up Kit 50 samples removes interfering material 1 100 yl Mini Dialysis Kit 50 samples 1 kDa cut off up to 250 ul Mini Dialysis Kit 50 samples 1 kDa cut off up to 2 ml Mini Dialysis Kit 50 samples 8 kDa cut off up to 250 ul Mini Dialysis Kit 50 samples 8 kDa cut off up to 2 ml Nuclease Mix 0 5 ml removes nucleic acids Albumin and IgG Removal Kit 10 samples removes albumin and IgG from human serum 2 D Quant Kit 500 assays quantitation of 1 50 ul up to 50 ug protein Vivaspin 25 pack exchanges buffer and concentrates sample 2 D Protein Extraction Buffer Trial Kit 6 x for 10 ml prepares high quality protein lysates illustra triplePrep Kit 50 preps isolates simultaneously DNA RNA and protein from the same sample 18 80 6429 60 AD 1 0 4 General sample preparation guidelines Keep the sample preparation strategy as simple as possible to avoid protein losses Additional sample preparation steps may improve the quality of the final 2 D result but at the possible expense of selective protein lo
211. one edge The electrode cable is not plugged in Cathodic buffer strip not in contact with the gel at one edge Ensure that all cables are properly connected Ensure that the cathodic buffer strip is centered and covers the entire width of the second dimension gel Dye front curves up smiles at both edges Inadequate cooling Ensure that the thermostatic circulator is connected to the Multiphor II unit and functioning correctly Dye front is irregular Some dye front irregularity results from the use of IPG Buffer and does not affect results Buffer strips or ExcelGel gels are old Ensure that the expiration dates on the buffer strips and ExcelGel gels have not elapsed Bubbles under the buffer strip Ensure that the buffer strips are placed firmly on the gel with no air bubbles trapped beneath them Bubbles under the Immobiline DryStrip gel Ensure that the Immobiline DryStrip gel is placed firmly on the gel with no air bubbles trapped underneath Stroke the plastic backing of the Immobiline DryStrip gel gently with a pair of forceps to remove trapped bubbles Buffer strip slides out from under the electrode Incorrect electrode placement Ensure that the electrodes are aligned over the center of the buffer strips before lowering the electrode holder 104 80 6429 60 AD 5 Visualizing and evaluating results 5 0 Visualizing results labeling and staining Most detection method
212. onto the glass plate The arrow indicates the direction of motion in applying the streak 3 Remove the protective plastic sheet from the gel Handling the gel only by the side support film margins hold it gel side down over the glass plate Ensure that it is oriented with the cathodic edge of the gel toward the cathodic edge of the cassette Align the right edge of the gel with the right edge of the side spacer of the glass plate side flex the gel downward slightly and lower it slowly toward the glass plate from right to left Take care that the bottom anodic edge of the gel is flush within 1 mm of the bottom anodic edge of the glass plate The protruding side support film margins but not the gel should rest on top of the side spacers 4 Remove bubbles and excess buffer Use the roller a separate accessory to press out any bubbles or liquid from between the gel and the glass Press firmly against the plastic support film with the roller and roll over the entire gel Fig 37 After rolling the gel should adhere firmly to the glass and resist further movement 5 Close the cassette Close the cassette snap the plastic frame to the glass plate Fig 38 and press the edges tightly together along the entire side of the cassette Ensure that the cassette is closed completely an incompletely closed cassette causes a strongly curved front 6 Repeat the procedure for each second dimension gel to be run 76 80 6429 60 AD Fig 3
213. ood while wearing a disposable dust mask Follow all local rules and regulations for handling and disposal of materials A Sample preparation solution with urea for 2 D electrophoresis 8 M urea 4 CHAPS 2 Pharmalyte or IPG buffer carrier ampholytes 40 mM DTT 25 ml Final concentration Amount Urea FW 60 06 8 M 12g CHAPSt 4 w v 10g Pharmalytet or IPG Buffers 2 v v 500 ul DTT FW 154 2 40 mM 154 mg Double distilled water to 25 ml 16 ml required If necessary the concentration of urea can be increased to 9 or 9 8 M t Other neutral or zwitterionic detergents may be used at concentrations up to 2 w v Examples include Triton X 100 NP 40 octyl glucoside and the alkylamidosulfobetaine detergents ASB 14 and ASB 16 Calbiochem Carrier ampholytes Pharmalyte or IPG buffer and DTT should be excluded from the sample extraction solution if the samples are to be labeled using 2 D DIGE See Ettan DIGE User Manual for details 5 Use IPG Buffer in the pH range corresponding to the pH range of the IEF separation to be performed or Pharmalyte in a pH range approximating the pH range of the IEF separation to be performed Store in 2 5 ml aliquots at 20 C Note Protease inhibitors may be added if necessary B Sample preparation solution with urea and thiourea for 2 D electrophoresis 7 M urea 2 M thiourea 4 CHAPS 2 Pharmalyte or IPG Buffer carrier ampholytes 40 mM DTT 25 ml
214. ophoresis System Multiphor II Electrophoresis Unit 18 1018 06 Multiphor II Buffer Strip Positioner 80 6442 90 IEF sample application pieces 200 pk 80 1129 46 Product Quantity Code No Power supplies EPS 3501 XL Power Supply 3500 V 400 mA 200 W 18 1130 05 EPS 2A200 Power Supply 200 V 2000 mA 200 W 80 6406 99 EPS 301 Power Supply 300 V 400 mA 80 W 18 1130 01 EPS 601 Power Supply 600 V 400 mA 100 W 18 1130 02 EPS 1001 Power Supply 1000 V 400 mA 100 W 18 1130 03 Thermostatic circulator MultiTemp IlI Thermostatic Circulator 115 V 18 1102 77 MultiTemp IlI Thermostatic Circulator 230 V 18 1102 78 ExcelGel SDS gels ExcelGel SDS 2 D Homogeneous 12 5 6 pk 17 6002 21 ExcelGel SDS Gradient XL 12 14 3 pk 17 1236 01 ExcelGel SDS Buffer Strips anode and cathode 6 each pk 17 1342 01 PlusOne chemicals and reagents Acrylamide PAGE acrylic acid lt 0 05 250g 17 1302 01 Acrylamide PAGE acrylic acid lt 0 05 1kg 17 1302 02 Acrylamide IEF acrylic acid lt 0 002 250g 17 1300 01 Acrylamide IEF acrylic acid lt 0 002 1kg 17 1300 02 Acrylamide IEF 40 solution 1 17 1301 01 Acrylamide PAGE 40 solution 1 17 1303 01 N N methylenebisacrylamide 259 17 1304 01 N N methylenebisacrylamide 100g 17 1304 02 N N methylenebisacrylamide 2 solution 1 17 1306 01 ReadySol IEF 40 T and 3 C 1 17 1310 01 Agarose NA 10g 17 0554 01 Glycine 500g 17 1323 01 Ammonium persulfate 25g 17 1311 01 TEMED 25 m
215. or 3 t is possible that the programmed maximum voltage will not be reached when using shorter Immobiline DryStrip gels or with samples having high conductivity The final step of focusing should be run in volt hours to ensure reproducibility from run to run The following protocols are suitable for first dimension isoelectric focusing of proteins run on Ettan IPGphor 3 Isoelectric Focusing Unit E7 cP G Q Preparative sample loads often increase the electroosmotic pumping of water Excess free water on the gel surface contributes to streaky results and should be absorbed with electrode pads This technique is standard when using the Ettan IPGphor 3 Manifold for standard Strip Holders this technique is described in section 2 7 The focusing times below are guidelines only based on well prepared samples Times may vary with the nature of the sample and how the sample is applied Using crude samples with high protein and salt content or using paper bridge loading the run time in total kiloVolt hours should be increased by 10 For Immobiline Dry Strip pH 6 2 7 5 6 9 6 11 and 7 11 NL loading the sample anodically in a sample cup is recommended For preparative sample loads with these basic strips paper bridge loading is recommended If using the Manifold and 18 and 24 cm strips the maximum voltage is 10 000 V With these two strip lengths and standard Strip Holders the maximum allowed voltage is 8000 V With all other strips an
216. or 3 Isoelectric Focusing System Fig 1 simplifies the first dimension separation with a system dedicated to IEF on Immobiline DryStrip gels Ettan IPGphor 3 consistently delivers speed and reproducibility and can handle high protein loads The system incorporates a safe high voltage up to 10 000 V depending on the DryStrip being used power supply and Peltier solid state temperature control 15 30 C Programmable parameters include rehydration temperature and duration IEF temperature and maximum current and the duration and voltage pattern of multiple steps for each separation In addition to the IEF unit key accessories include Ettan IPGphor Manifold Strip Holders and IPGbox Integral Ettan IPGphor 3 Control Software provides greater control in IEF runs it can be used to control up to four Ettan IPGphor 3 units simultaneously each running a different set of parameters These accessories are discussed in detail in section 2 3 cr For gradients at the upper and lower ends of the pH scale as well as for very high protein loads on narrow pH range gradient strips Ettan IPGphor 3 Manifold is employed for IEF using 7 11 13 18 and 24 cm Immobiline DryStrip gels Samples can be loaded onto IPG strips using sample cups Ettan IPGphor 3 IPGbox or paper bridges Sections 2 3 2 5 discuss these options The versatile Multiphor II Electrophoresis System Fig 7 can be used to perform several different electrophoresis techniques An advantage
217. or use in protein transfers It has higher mechanical strength than unsupported nitrocellulose and a protein binding capacity of 125 ug cm Hybond P is chemically stable allowing the use of a range of solvents for rapid de staining EF The plastic backing on DALT and ExcelGel precast gels is removed with the Film Remover prior to electrotransfer see ordering information 5 2 Evaluating results n theory the analysis of up to 15 000 proteins should be possible in one gel in practice however 5000 detected protein spots means a very good separation Evaluating high resolution 2 D gels by a manual comparison of two gels is not always possible In large studies with patterns containing several thousand spots it may be almost impossible o detect the appearance of a few new spots or the disappearance of single spots Image collection hardware and image evaluation software are necessary to detect these differences as well as to obtain maximum information from he gel patterns mageMaster 2D Platinum and DeCyder 2 D Differential Analysis software together with ImageScanner and or Typhoon multicolor fluorescence and phosphor image scanner comprise a system that allows the user to capture store evaluate and present information contained in 2 D gels e ImageScanner II desktop instrument captures optical information in the visible wavelength range over a range from 0 to more than 3 4 O D in reflection or transmission mode It scans 20 x 2
218. ormation relating to DeCyder 2 D Differential Analysis Software please refer to the Ettan DIGE user manual e Extended Data Analysis EDA Multivariate analysis of data from several BVA workspaces 6 3 8 Further analysis of protein spots Ettan DIGE system is fully compatible with mass spectrometry analysis and has been fully integrated into the Ettan proteomics platform DeCyder 2 D software will generate a pick list of spots of interest that can be exported directly into Ettan Spot Picker or Ettan Spot Handling Workstation Protein spots are automatically picked from the glass backed gel For backing of gels to glass see appendix V Although spots can be picked directly from post stained analytical gels where possible preparative scale gels provide more material for analysis by mass spectrometry A preparative gel post stained can be matched to previous analytical gels by DeCyder 2 D Differential Analysis Software See also section 5 4 80 6429 60 AD 123 6 4 Troubleshooting 2 D DIGE For troubleshooting 2 D DIGE results please refer to the Ettan DIGE user manual 124 80 6429 60 AD 7 Troubleshooting Table 44 lists problems that may be encountered in 2 D electrophoresis results describes the possible causes and suggests ways to prevent problems in future experiments For troubleshooting problems encountered during the various steps of the 2 D process refer to the following e Table 20 page 70 Troubleshooting first dim
219. ortions thereof is manufactured under an exclusive license from Carnegie Mellon University under US patent numbers 5 569 587 5 627 027 and equivalent patents in other countries The purchase of CyDye DIGE Fluors includes a limited license to use the CyDye DIGE Fluors for internal research and development but not for any commercial purposes A license to use the CyDye DIGE Fluors for commercial purposes is subject to a separate license agreement with GE Healthcare CyDye This product or portions thereof is manufactured under an exclusive license from Carnegie Mellon University under US patent number 5 268 486 and equivalent patents in the US and other countries The purchase of CyDye products includes a limited license to use the CyDye products for internal research and development but not for any commercial purposes A license to use the CyDye products for commercial purposes is subject to a separate license agreement with GE Healthcare Commercial use shall include 1 Sale lease license or other transfer of the material or any material derived or produced from it 2 Sale lease license or other grant of rights to use this material or any material derived or produced from it 3 Use of this material to perform services for a fee for third parties including contract research and drug screening If you require a commercial license to use this material and do not have one return this material unopened to GE Healthcare Bio Sciences AB Bjorkga
220. oteins or just a particular subcellular fraction Gentle and vigorous lysis methods are discussed in sections 1 1 1 and 1 1 2 respectively A protocol for grinding cells using Sample Grinding Kit can be found in section 1 1 3 Proteases may be liberated upon cell disruption Proteolysis greatly complicates analysis of the 2 D gel result thus the protein sample should be protected from proteolysis during cell disruption and subsequent preparation Protease inhibition is discussed in section 1 2 Section 1 2 1 provides protocols for use of Protease Inhibitor Mix If only a subset of the proteins in a tissue or cell type is of interest fractionation can be employed during sample preparation If proteins from one particular subcellular compartment e g nuclei mitochondria plasma membrane are desired the organelle of interest can be purified by differential centrifugation or other means prior to solubilization of proteins for 2 D electrophoresis The sample can also be fractionated by solubility under different extraction conditions prior to 2 D electrophoresis see references 9 13 for experimental conditions 1 0 2 Precipitation and removal of interfering substances In whole cell lysates proteins are present in a wide dynamic range of concentrations In such a situation abundant proteins may mask identification of less abundant proteins of interest An effective proteome analysis will naturally require separation of abundant proteins and enrichment
221. oteomics 2 727 732 2002 Bradford M M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding Anal Biochem 72 248 254 1976 Lowry O H et al Protein measurement with the folin phenol reagent J Biol Chem 193 265 275 1951 Smith P K et al Measurement of protein using bicinchoninic acid Anal Biochem 150 76 85 1985 Goshey and Nedkov P Extending the range of application of the biuret reaction quantitative determination of insoluble proteins Anal Biochem 95 340 343 1979 Bjellqvist B et al Micropreparative two dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins Electrophoresis 14 1375 1378 1993 Sanchez J C et al Improved and simplified in gel sample application using reswelling of dry immobilized pH gradients Electrophoresis 18 324 327 1997 Rabilloud T et al Sample application by in gel rehydration improves the resolution of two dimensional electrophoresis with immobilized pH gradients in the first dimension Electrophoresis 15 1552 1558 1994 Westermeier R Electrophoresis in Practice 3rd Ed Wiley VCH Verlag GmbH Weinheim 2001 Westermeier R and Naven T Proteomics in Practice A Laboratory Manual of Proteome Analysis Wiley VCH Verlag GmbH Weinheim 2002 Sabounchi Schutt F et al An Immobiline DryStrip application method enabling high
222. oth systems are designed for simplified assembly and rapid electrophoresis of the second dimension gel Ettan DALTsix system accepts up to six large second dimension gels 26 x 20 cm Fig 2 on page 11 Ettan DALTtwelve system can handle up to 12 large gels Fig 3 on page 12 When running fewer gels unused slots are filled with blank cassette inserts Safety interlocks prevent the application of power to the separation unit unless the lid is properly closed Both units recirculate the buffer so that even gel temperatures are maintained during electrophoresis Most of the steps are common between Ettan DALTsix and Ettan DALTtwelve systems and thus the protocols presented below apply to both Where there are differences in two instances preparing the system for electrophoresis and inserting gels into the system alternate protocols are presented under the same section number Power supply and temperature control unit The modular Ettan DALTsix system requires an external power supply and thermostatic circulator to control the buffer temperature A power supply capable of 100 W constant power output such as the EPS 601 is recommended for the fastest separation time For temperature control a circulating water bath such as the MultiTemp III should be used The operating temperature range of Ettan DALTsix system is 4 40 C Ettan DALTtwelve system is controlled from the Power Supply Control Unit The unit supplies a maximum power output of 200 W
223. ounced streaking on 2 D gels caused by low molecular weight contaminants Fig 10 The kit contains dialysis tubes each of which consists of a sample tube with a cap that is fitted with a dialysis membrane Sample is easily and quantitatively transferred into and out of the tube by pipetting pH 3 10NL gt pH 3 10NL gt settee tet RS Undialyzed Dialyzed with Mini Dialysis Kit Fig 10 Effect of dialysis on 2 D resolution Sample E coli protein extracted with 15 mM NaCl 8 M urea 0 5 Pharmalyte pH 3 10 2 CHAPS Dialysis Mini Dialysis Kit 8 kDa 250 ul 17 h against 8 M urea First dimension Approximately 400 ug E coli protein 13 cm Immobiline DryStrip pH 3 10 NL Ettan IPGphor Isoelectric Focusing System 32 kVh Second dimension SDS PAGE 12 5 SE 600 vertical electrophoresis system 16 x 16 cm gel Stain Colloidal Coomassie G 250 Q dialysis membrane float Fig 11 Schematic of the method used in Mini Dialysis Kit a Cap with dialysis membrane conical inner sample tube b Introduce sample screw on cap and slide tube into float c Invert and dialyze while stirring d Spin briefly to collect sample The capped tube is inverted in a stirred beaker containing the solution against which the sample is to be dialyzed Salts and molecules smaller than the molecular weight cut off of the dialysis membrane are effectively exchanged through the membrane Followin
224. oupled with the 24 cm or 18 cm Immobiline DryStrip gel is the best choice Using the buffer strip positioner helps to get optimal results good reproducibility is achieved because of standardized placement of Immobiline DryStrip gels and buffer strips and a straight run because the gel surface is covered A flatbed second dimension system is not recommended if the first dimension has been run on a pH 6 9 6 11 or 7 11 NL Immobiline DryStrip gel Protocol 1 Equilibrate the Immobiline DryStrip gels During the preparation of the ExcelGel SDS gel equilibrate the Immobiline DryStrip gels as described in section 3 1 2 2 Prepare the Multiphor II Electrophoresis System Set the temperature on the MultiTemp III Thermostatic Circulator to 15 C Pipette 2 5 3 0 ml of Immobiline DryStrip Cover Fluid onto the Multiphor II cooling plate 3 Position the ExcelGel SDS gel Remove the gel from the foil package by cutting away the edges of the package A notch at the lower left hand corner of the film identifies the anodic side Note The gel is cast onto a plastic support film and does not cover the film entirely Both gel types contain a stacking gel zone with 5 acrylamide Markings on the plastic cover indicate the direction of electrophoresis Orient the gel according to these markings remove the cover and place the gel on the cooling plate The cathodic edge of the ExcelGel SDS must align and make uniform contact with the cathodic edge of the g
225. ous swirling of the resin bottle before the removal of each aliquot is recommended to ensure the resin remains in suspension Remove an aliquot quickly using a wide mouthed pipette tip 80 6429 60 AD 35 ETF During serum resin incubation ensure that the resin is kept in suspension by adequate mixing on a rotary shaker Ensure that centrifugation is performed at the correct g force and for the required time This allows all the liquid to be eluted from the resin following sample treatment The resin should appear dry following centrifugation EFT Use of the 2 D Clean Up Kit makes acetone precipitation unnecessary However if performing an acetone precipitation do not over dry acetone precipitated protein pellets Guidelines for serum sample loading with the Albumin IgG Removal Kit are listed in Table 12 The values are those seen when a typical human serum sample is treated using the Albumin and IgG Removal Kit Human serum samples contain widely varying levels of albumin and IgG and the information below should be used for guidance only Table 12 Typical levels for removal of albumin and IgG Human serum sample volume Typical level of albumin removed Typical level of IgG removed 15 ul gt 95 gt 90 30 ul gt 80 gt 80 45 ul gt 60 70 gt 70 1 Pipette 15 ul of human serum into a sample tube with lid Tubes to be used for sample incubation should be of adequate size to allow good mixing of the resin serum
226. ow all local safety rules and regulations including for disposal Quick guide for finding information on gel casting for DALTsix and DALTtwelve electrophoresis systems To find gel casting information quickly refer to Table 25 for gel volumes required Table 26 for single percentage gel recipes and Table 27 for gradient gel recipes The instructions provided below for the preparation of vertical SDS polyacrylamide gels employ the Tris glycine system of Laemmli 78 Vertical second dimension gels are most conveniently cast several at a time in a multiple gel caster see ordering information For assembly of the gel cassette refer to the relevant user manual 80 80 6429 60 AD Protocol 1 Select the gel percentage a Single percentage gel versus gradient gel When a gradient gel is used the overall separation interval is wider and the linear separation interval is larger In addition sharper bands result because the decreasing pore size functions to minimize diffusion However a gradient gel requires more skill to cast For detailed instructions on gradient preparation see the user manual for the relevant electrophoresis unit and multiple gel caster Single percentage gels offer better resolution for a particular M window A commonly used second dimension gel for 2 D electrophoresis is a homogeneous gel containing 12 5 total acrylamide Note Stacking gels are not necessary for vertical 2 D gels b Whether single percentage or gra
227. owing exceptions 1 Prepare twice the volume of fixing solution as indicated in the kit instructions i e 500 ml per gel rather than 250 ml 2 Prepare the developing solution with twice the volume of formaldehyde solution as indicated in the kit instructions i e 100 ul per 250 ml rather than 50 ul per 250 ml 3 Stain the gels according to the following protocol Step Solutions Amount Time Fixation Ethanol 200 ml 2 x 60 min Acetic acid glacial 50 ml Make up to 500 ml with distilled water Sensitizing Ethanol 75 ml 60 min Glutardialdehydet 25 w v 1 25 ml Sodium thiosulfate 5 w v 10 ml Sodium acetate 17 g 1 packet Make up to 250 ml with distilled water Washing Distilled water 5x8min Silver reaction Silver nitrate solution 2 5 w v 25 ml 60 min Formaldehydet 37 w v 0 1 ml Make up to 250 ml with distilled water Washing Distilled water 4x1min Developing Sodium carbonate 6 25 g 1 packet 5 min Formaldehyde 37 100 ul Make up to 250 ml with distilled water Stir vigorously to dissolve sodium carbonate Stop Na EDTA H 0 3 65 g 1 packet 45 min Make up to 250 ml with distilled water Washing Distilled water 2 x 30 min Preservationt Glycerol supplied at 87 final conc 4 12 5 mls 20 min Made up to 250 ml with distilled water OR Ethanol 10 v v tt 25 ml Made up to 250 ml with distilled water xk The first fixation may be prolonged up to 3 days if desired By omitting glu
228. p pH 3 10 NL to distribute the proteins more evenly over the strip This is especially helpful when analyzing complex samples like serum cerebrospinal fluid extracts from E coli and yeasts e Combine pH 3 7 and pH 6 11 or pH 3 7 and pH 6 9 or select from pH 3 5 6 NL 5 3 6 5 6 2 7 5 or 7 11 NL when more detail is required Of these the two NL strips pH 3 5 6 NL and 7 11 NL are nonlinear at the extreme ends of the pH scale allowing a more even distribution of proteins over the gel length and maximized resolution The pH 3 5 6 NL 5 3 6 5 6 2 7 5 and 7 11 NL strips provide optimal overlaps and approximately the same number of proteins is separated in each pH interval Higher sample loading capacity of medium range gels makes protein identification easier Q Er Use narrow pH range strips 1 pH unit to closely study proteins in the regions of interest Narrow gradients of 1 pH unit allow higher resolution in depth study of proteins separating in these regions and increased loading capacity Several milligrams of protein extract can be analyzed when using rehydration loading including protein samples in the rehydration solution which simplifies identification and characterization of spots in the 2 D map Note To increase the stability of the pH gradient in Immobiline DryStrip pH 7 11 NL during production the buffering capacity is enhanced at the most basic end by the introduction of a proprietary arginine derivative guanidy
229. p and Hold 500 2 00 5 00 1 0 2 Gradient 1000 2 00 15 3a Gradientt 8000 3 00 13 5 4a Step and Holdt 8000 9 10 10 30 74 0 84 0 3b Gradient 10000 3 00 16 5 4b Step and Hold 10000 7 05 8 05 71 0 81 0 Total 90 0 100 0 53 65 1 Step and Hold 500 2 00 5 00 1 0 6 2 7 5 2 Gradient 1000 2 00 15 3a Gradientt 8000 3 00 13 5 4a Step and Holdt 8000 9 19 10 30 94 0 114 0 3b Gradientt 10000 3 00 16 5 4b Step and Holdt 10000 9 05 11 05 91 0 111 0 Total 110 0 130 0 When a more convenient overnight run of 15 to 17 h is desired the time in step 1 can be prolonged to up to recommended values in brackets Using this option step 4 can be reduced with the added kVh in step 1 to reach the specified total kVh t Follow steps 1 2 3a and 4a when using IPGphor Regular Strip Holder or Cup Loading Strip Holder Follow steps 1 2 3b and 4b when using IPGphor Cup Loading Manifold CF If using regular Strip Holders active rehydration can be performed if sample is included by adding an extra step at the beginning of the protocol e g Voltage mode 1 Step and Hold Voltage 30 Step duration 10 00 kVolt hours 0 3 kVh 2 8 3 Preservation of focused Immobiline DryStrip gels After IEF is complete proceed to the second dimension separation immediately or store the Immobiline DryStrip gels at 60 C or below This can be conveniently done by placing the strips between plastic sheets as suggested by Gorg et al 3 or on glass plates covered in pla
230. patocytes biochemical immunochemical and two dimensional gel electrophoresis characterization of cytoskeletal and noncytoskeletal compartments Electrophoresis 15 265 277 1994 Taylor R S et al Proteomics of rat liver Golgi complex minor proteins are identified through sequential fractionation Electrophoresis 21 3441 3459 2000 Deutscher M P ed Guide to protein purification Methods Enzymol 182 1 894 1990 Dunn M J and Corbett J M 2 dimensional polyacrylamide gel electrophoresis Methods Enzymol 271 177 203 1996 Rabilloud T Solubilization of proteins for electrophoretic analysis Electrophoresis 17 813 829 1996 Rabilloud T et al Improvement of the solubilization of proteins in two dimensional electrophoresis with immobilized pH gradients Electrophoresis 18 307 316 1997 Scopes R K Making an Extract in Protein purification Principles and practice 2nd Ed Springer Verlag New York 1987 Dignam J D Preparation of extracts from higher eukaryotes Methods Enzymol 182 194 203 1990 Toda T et al Detection of thymopoietin responsive proteins in nude mouse spleen cells by two dimensional polyacrylamide gel electrophoresis and image processing Electrophoresis 15 984 987 1994 Sanchez J C et al Inside SWISS 2D PAGE database Electrophoresis 16 1131 1151 1995 Portig I et al Identification of stress proteins in endothelial cells Electrophoresis 17 803 808
231. ple Total Protein Stain For clarity the gel images show pH 3 8 where most of the proteins are present The expanded region of the gel stained with Sypro Ruby gel A and resulting 3 D plot demonstrate the drawbacks associated with speckling Staining with Deep Purple Total Protein Stain gel B eliminates speckling and improves spot clarity which allows more accurate spot detection and protein identification First dimension pH 3 10 NL 24 cm Immobiline DryStrip strip run on Ettan IPGphor 3 IEF System second dimension 12 5 SDS electrophoresis gel run on Ettan DALTtwelve electrophoresis system Scanned using Typhoon 9410 Variable Mode Imager Full experimental details can be found in application note 18 1177 44 cr The plastic backing on precast gels can pose a problem of high background when fluorescent staining and labeling techniques are used 5 0 1 Automating processing and preserving the gel Processor Plus automates multistep staining processes for increased convenience and reproducibility Automated protocols have been developed to use PlusOne Silver Staining Kit Protein to silver stain proteins in SDS gels This convenient adaptation gives reproducible results and sensitivity below 1 ng per spot for most proteins With a modification for subsequent mass spectrometry detection down to approximately 5 ng per spot can be achieved 84 For further information regarding methodology please refer to the Processor Plus Protocol Guide see addit
232. prevent dehydration of rehydrated Immobiline DryStrip gels Immobiline DryStrips turn white and opaque after focusing mmobiline DryStrip gels dried out during IEF Always apply recommended amount of Immobiline DryStrip Cover Fluid to prevent dehydration of rehydrated Immobiline DryStrip gels Immobiline DryStrip Cover Fluid overflows from Manifold Excess cover fluid added Do not add more than the recommended volume Ensure that the outside rim of the tray does not have any oil on it 80 6429 60 AD 69 70 80 6429 60 AD 3 Second dimension SDS PAGE using vertical electrophoresis systems 3 0 Overview After IEF the second dimension SDS polyacrylamide gel electrophoresis SDS PAGE can be performed on various vertical or flatbed systems depending on factors such as those discussed in Equipment Choices on pages 14 16 SDS PAGE consists of four steps 1 Preparing the system for second dimension electrophoresis N Equilibrating the Immobiline DryStrip gells in SDS equilibration buffer W Placing the equilibrated Immobiline DryStrip gel on the SDS gel Electrophoresis The equilibration step is described first because it is a protocol common to all electrophoresis systems described in this handbook Gel preparation Immobiline DryStrip gel placement and electrophoresis protocols on the other hand are specific to the orientation of the gel Sections 3 3 and 3 4 describe these protocols as t
233. previously provided for DeStreak Rehydration Solution 2 6 3 Preparation of other rehydration solutions Typical compositions of rehydration solutions are given in appendix I solutions C and D 2 7 Immobiline DryStrip Gel rehydration using accessories This section includes protocols for use of the Strip Holder IPGbox and Manifold It covers the following scenarios e Using Strip Holders for rehydration loading sample included or sample loading after gel rehydration e Using IPGbox prior to using the Manifold sample added prior to reswelling or after reswelling using cup or paper bridge loading For rehydration sample loading the Immobiline DryStrip gel must be rehydrated in the Immobiline DryStrip IPGbox or in the standard Strip Holder Mix the sample with rehydration solution see section 2 5 for recommended sample loads When the Immobiline DryStrip gels are rehydrated with the sample proteins sample cups are not used This approach is referred to as passive rehydration In some cases rehydration under voltage referred to as active rehydration might be preferred Rehydration under low voltage 20 120 V facilitates the entry of high molecular weight proteins 70 Active rehydration is possible only in the Strip Holder Large sample volumes and large protein amounts can be applied using paper bridge loading Manifold only For example for basic proteins a paper pad paper bridge is soaked with sample and placed between the ano
234. proves the resolution of lower abundance proteins in two ways by enabling visualization of proteins that co migrate with albumin and IgG and by removal of a large portion of the total serum protein which allows an increase in the protein load of the low abundant proteins Affinity resins selectively remove these contaminants from human serum continues on following page 30 80 6429 60 AD Table 11 Contaminants that affect 2 D results continued Contaminant Reason for removal Removal techniques lonic detergent lonic detergent usually SDS is often used during protein extraction and solubilization but can strongly interfere with IEF SDS forms complexes with proteins and the resulting negatively charged complex will not focus unless the SDS is removed or sequestered Dilute the SDS containing sample into a rehydration solution containing a zwitterionic or nonionic detergent CHAPS Triton X 100 or Nonidet P 40 NP 40 so the final concentration of SDS is 0 25 or lower and of SDS is 0 25 or lower and the ratio of the other detergent to SDS is at least 8 1 27 Acetone precipitation of the protein will partially remove SDS Precipitation at room temperature will maximize removal of SDS but protein precipitation is more complete at 20 C 45 Nucleic acids DNA RNA Nucleic acids increase sample viscosity and cause background smears High molecular weight nucleic acids can clog ge
235. proximately 1 2 of lysine residues As a result CyDye DIGE Fluor minimal dyes will label only a small proportion of each protein in a sample hence the expression minimal labeling The lysine amino acid in proteins carries an intrinsic positive charge which when a CyDye DIGE Fluor minimal dye is coupled to the lysine replaces the lysine s single positive charge with its own ensuring that the pl of the protein does not change When coupled to the protein CyDye DIGE Fluor minimal dyes add approximately M 500 to the protein s mass However proteins should not be picked using the CyDye labeled gel image as a positional reference due to slight migration differences between the unlabeled and labeled proteins These differences are due to the addition of a single CyDye molecule to the labeled protein which decreases the mobility of the protein with respect to unlabeled protein This effect is more marked for lower molecular weight proteins To ensure that the majority of unlabeled protein is picked and therefore that sufficient protein is available for identification by mass spectrometry any gel for picking usually a preparative gel can be post stained with a suitable stain such as Deep Purple see section 5 0 and appendix IV The resulting gel is matched to the analytical set of DIGE gels within the DeCyder 2 D Differential Analysis Software protein linker pHss Lysine NHS ester bi reactive group o N H O Fig 61 Sch
236. r 2D 7 1 SPN 1 user license upgrade from 2D 7 0 1 28 9763 21 DeCyder 2D 7 1 SPN Software Package 1 28 9757 78 Installation Guide Getting Started Guide and DeCyder 2D DVD case with installation disc packaged in DeCyder box 158 80 6429 60 AD Recommended additional consumables Sulfobetaines PefaBloc DMF N N dimethylformamide 99 8 anhydrous Crew Wipes L lysine Molecular Sieves 4A Decon 90 22 705 6 223681 0 L 5626 M2635 cln 010 Calbiochem Merck Sigma Aldrich Sigma Aldrich Sigma Aldrich Sigma Aldrich 010 M J Patterson Scientific Ltd 80 6429 60 AD 159 For local office contact information please visit www gelifesciences com contact www gelifesciences com protein purification GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden imagination at work GE imagination at work and GE monogram are trademarks of General Electric Company Cy CyDye DeCyder Deep Purple ECL Ettan ExcelGel Hybond illustra ImageMaster ImageQuant ImageScanner Immobiline PGphor Multiphor MultiTemp Personal Densitometer Pharmalyte Rainbow Scierra and Typhoon are trademarks of GE Healthcare companies 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE technology is covered by US patent numbers 6 043 025 6 127 134 and 6 426 190 and equivalent patents and patent applications in other countries and exclusively licensed from Carnegie Mellon University CyDye this product or p
237. r electrophoresis remove gels from their gel cassettes in preparation for staining or blotting Notch or mark each gel at the upper corner nearest the or end of the Immobiline DryStrip gel to identify the acidic end of the first dimension separation Table 33 Recommended electrophoresis conditions for second dimension vertical gels Step Current Approximate run mA gel duration h min miniVE and SE 260 1 0 mm thick gels 1 10 0 15 2 20 1 30 1 5 mm thick gels 1 15 0 15 2 30 1 30 SE 600 Ruby 1 0 mm thick gels 1 10 0 15 2 50 20 2 00 5 00 1 5 mm thick gels 1 15 0 15 2 60 30 2 00 5 00 The time shown is approximate Stop electrophoresis when the dye front is 1 mm from the bottom of the gel v If running at the higher currents cooling is highly recommended 3 5 Troubleshooting Table 34 lists possible problems that might be encountered during vertical SDS PAGE and how to solve them Table 34 Troubleshooting vertical second dimension SDS PAGE Symptom Possible cause Remedy No current at Insufficient volume of buffer in upper Ensure that both reservoirs contain enough SDS start of run or lower reservoir electrophoresis buffer to contact both upper and lower electrode wires Check for leaks Second dimension SDS electrophoresis buffer prepared Make fresh solutions separation proceeds incorrectly or resolving gel buffer too slowly prepared incorrectly Current leakage
238. r to overnight electrophoresis Modular system Precast gels with stable buffer system available cast on film support Ettan DALT Gel 12 5 25 5 x 19 6 cm 1 mm thickness Large format gels for highest resolution and maximum protein load Medium throughput up to six gels simultaneously Best for 18 and 24 cm IPG strips Buffer volume approximately 5 for six gels 80 6429 60AD 9 Ettan DALTtwelve Large Vertical System up to twelve 26 x 20 cm gels Choice Factors e Four hour to overnight electrophoresis e Integrated system with very efficient Peltier temperature control e Precast gels with stable buffer system available cast on film support Ettan DALT Gel 12 5 25 5 x 19 6 cm 1 mm thickness e Large format gels for highest resolution and maximum protein load Fig 3 Ettan DALTtwelve Large Vertical System High throughput up to 12 gels simultaneously Best for 18 and 24 cm IPG strips Buffer volume approximately 10 for 12 gels miniVE and SE 260 Mini Vertical one or two 8 x 7 or 9 5 cm gels Choice Factors e Rapid 1 2 h electrophoresis e Best for 7 cm IPG strips e Ideal when quick profiling is required or when the protein pattern is relatively simple Fig 4 miniVE Vertical Eelctrophoresis System Fig 5 SE 260 10 80 6429 60 AD SE 600 Ruby standard vertical one to four 14 x 16 cm gels Choice Factors e Electrophoresis in 2 5 h e Intermediate separation 16 cm
239. r wash solution in the appropriate volumes must be used Do not dilute the stain beyond 1 200 for gels and 1 400 for blots as this will result in reduced intensity and sensitivity cP EF Do not reuse the stain solution as this may result in a significant loss of sensitivity If the whole staining process takes more than 8 hours process gel containers should be covered to exclude light and agitated gently on a mixer platform Solution and reagents required A Fixation Acidification solution for gels 15 v v ethanol 1 citric acid v v in water approx pH 2 3 Add 150 ml of 99 9 ethanol and 10 g citric acid to 850 ml water check pH Fixing solution can be stored at room temperature for up to six months Note Ethanol 15 v v in water can be replaced by methanol 30 v v in water Citric acid 1 v v in water can be replaced by acetic acid 7 5 v v in water The concentration of acetic acid should not exceed 10 B Working stain solution for gels and blots 100 mM sodium borate pH 10 5 in water Dissolve 6 2 g boric acid in 800 ml water and adjust pH to 10 5 with NaOH then make to 1 This solution should be made fresh at the time of use by adding 1 part Deep Purple to 200 parts borate buffer for gels or 1 part Deep Purple to 400 parts of borate buffer for blots Boric acid NaOH buffer may be prepared in advance and can be stored for up to 6 months C Wash solution for gels 15 v v ethanol in water Add 150 ml ethano
240. rb excess water Paper bridges and electrode pads are cut from 1 mm thick CleanGel electrode strips see ordering information to a size of 15 x 25 mm and with an arrowhead as shown in Figure 52 The rehydrated Immobiline DryStrip gel is positioned directly on the glass bottom of the Immobiline DryStrip tray Up to four Immobiline DryStrip gels can be run simultaneously on the Multiphor II Electrophoresis System The arrow headed paper to which 375 ul sample solution has been added is then positioned at the anodic or the cathodic end of the Immobiline DryStrip gel To hold the paper bridge and Immobiline DryStrip gel in place press a sample cup positioned on the sample cup bar down on top of the arrowhead A solution containing up to 10 mg protein in 850 ul sample solution applied to a 15 x 50 paper bridge can be loaded on an 18 cm long narrow pH range Immobiline DryStrip gel under favorable conditions 74 The application point anodic or cathodic is of key importance for obtaining good results 80 6429 60 AD 95 IPG Pressure from Paper Electrode Electrode strip sample cup bridge strip Fig 52 Setup for sample application via a paper bridge 4 1 5 IEF guidelines for Multiphor II Electrophoresis System IEF using the Multiphor II Electrophoresis System is conducted at high voltages up to 3500 V and very low currents typically less than 1 mA due to the low ionic strength within Immobiline DryStrip gels During IEF the current dec
241. reases while the voltage increases as proteins and other charged components migrate to their equilibrium positions In a typical IEF protocol voltage is gradually increased to the final desired focusing voltage which is held for several hours or more With cup loading a low initial voltage minimizes sample aggregation and generally allows the parallel separation of samples with differing salt concentrations The main factors determining the required volt hours Vh are the length of the Immobiline DryStrip gels and the pH gradient used Sample composition rehydration solution composition and sample application mode influence the required volt hours Table 37 gives volt hour values suitable for most samples with rehydration loading or anodic cup loading cr Cathodic sample application on wide range gradients pH 3 10 requires considerably longer focusing times than those stated in Table 37 especially if SDS containing samples are used As an example an SDS solubilized serum protein sample applied at the cathodic end of a pH 3 10 NL gradient requires Volt hours in excess of 2 2 5 fold of that stated in Table 37 75 ET Salt and buffer ions in the sample can require an increase of the time for phase 2 compared with the values given in Table 37 particularly when cup loading is used High ion concentrations in the sample can also require an increase of the total Volt hour requirement as these ions have to be transported to the ends of the Immobiline
242. reposition the tubes in the microcentrifuge with the cap hinges and pellets facing outward Centrifuge the tubes briefly to bring any remaining liquid to the bottom of the tubes Use a pipette to remove the remaining supernatant There should be no visible liquid remaining in the tubes 7 Without disturbing the pellet layer 40 ul of co precipitant on top of each pellet Incubate the tubes on ice for 5 min 8 Carefully reposition the tubes in the centrifuge with the cap hinges facing outward Centrifuge for 5 min Use a pipette to remove the supernatant 9 Pipette 25 ul of distilled or deionized water on top of each pellet Vortex each tube for 5 10 s The pellet should disperse but not dissolve in the water 80 6429 60 AD 27 0 Add 1 ml of wash buffer prechilled for at least 1 h at 20 C and 5 ul of wash additive to each tube Vortex until the pellets are fully dispersed Note The protein pellet will not dissolve in the wash buffer Fee Incubate the tubes at 20 C for at least 30 min Vortex for 20 30 s once every 10 min At this stage the tubes can be stored at 20 C for up to one week with minimal protein degradation or modification N Centrifuge the tubes at maximum speed at least 12 000 x g for 5 min W Carefully remove and discard the supernatant A white pellet should be visible Allow the pellet to air dry briefly no more than 5 min Coe Do not over dry the pellet If it becomes too dry it will b
243. rid on the cooling plate Avoid trapping bubbles between the gel and the cooling plate Avoid getting Immobiline DryStrip Cover Fluid on the gel surface as this may cause the buffer strips to slide during electrophoresis Separation quality is improved if the gel surface is allowed to dry uncovered for approximately 5 min before proceeding 4 Place the Multiphor II Buffer Strip Positioner The pegs protruding from the bottom of the positioner should be in contact with the shorter sides of the cooling plate Match the cathode and anode symbols on the positioner to the cathode and anode symbols on the cooling plate Slide the positioner so that the cathodic edge of the gel bisects the slot at position 1 see instructions provided with Multiphor II Buffer Strip Positioner Lock the positioner in place by turning the gray locking cam until the positioner cannot be moved 80 6429 60 AD 101 5 Position the cathodic buffer strip Carefully peel back the foil on the colorless cathodic ExcelGel SDS buffer strip Place the buffer strip with the smooth narrow face downward Align the buffer strip with the edge of the slot at position 1 and place it in the slot Fig 53 If the buffer strip breaks piece it together on the gel Vinyl gloves tend to stick less to the buffer strips than other types of plastic gloves If sticking persists dampen the gloves with distilled water or a 5 SDS solution 6 Position the anodic buffer strip Care
244. rips to the Ettan IPGphor 3 Manifold Place them face up in the tray with the anodic end of the strip resting on the appropriate mark etched on the bottom of the Manifold track 4 Seat cups in track if cup loading Place a strip of cups in the appropriate position The convenient seating tool enables you to push the cups down so that they are properly seated at the bottom of the track 5 Moisten and place electrode pads Wet the precut electrode pads with 150 ul deionized water and place the pads on the ends of the IPG Strips 6 Position electrode assembly Slide an electrode assembly over the top of all the pads Swivel the cams into the position under the external lip of the Manifold to seat the electrode in place 7 Load and cover samples Load the samples into the sample cups up to a maximum of 150 pl Check to make sure that the samples are completely covered with DryStrip Cover Fluid 8 Set program parameters and run Close the Ettan IPGphor 3 cover Select program and enter desired run parameters and begin the run 2 3 3 IPGbox IPGbox and IPGbox Kit are tools for enhancing the reswelling of GE Healthcare s precast Immobiline DryStrip Gels It provides a convenient method for rehydrating up to twelve precast IPG strips 7 to 24 cm at a time Individual slots in the Reswell Trays allow rehydration of individual IPG strips in a minimum volume of solution The IPGbox includes a lid that prote
245. ropriate rehydration solution for the sample will depend on its specific protein solubility requirements A typical solution generally contains urea nonionic or zwitterionic detergent DeStreak Reagent or DTT the appropriate Pharmalyte or IPG Buffer all available from GE Healthcare and a tracking dye The sample may also be included The role of each component is described below as well as the recommended concentration range Urea solubilizes and denatures proteins unfolding them to expose internal ionizable amino acids Commonly 8 M urea is used but the concentration can be increased to 9 or 9 8 M if necessary for complete sample solubilization Thiourea in addition to urea can be used to further improve protein solubilization particularly for hydrophobic proteins 10 16 55 57 When using both the recommended concentration of urea is 7 M and that of thiourea 2 M Detergent solubilizes hydrophobic proteins and minimizes protein aggregation The detergent must have zero net charge use only nonionic or zwitterionic detergents CHAPS Triton X 100 or NP 40 in the range of 0 5 to 4 are most commonly used DeStreak Reagent overcomes the problems of streaking that commonly occur due to reoxidation when running gels that contain basic regions above pH 7 The reagent stabilizes thiol groups such as disulfides thus reducing streaking and extra spots caused by various oxidation stages of proteins 62 See section 2 6 2 for more information an
246. rth M J Prevention of unwanted proteolysis in Proteolytic Enzymes A Practical Approach Beynon R J and Bond J S eds IRL Press Oxford pp 105 124 1989 Salvesen G and Nagase H Inhibition of proteolytic enzymes in Proteolytic Enzymes A Practical Approach Beynon R J and Bond J S eds IRL Press Oxford pp 83 104 1989 Hurkman W J and Tanaka C K Solubilization of plant membrane proteins for analysis by two dimensional gel electrophoresis Plant Physiol 81 802 806 1986 Granier F Extraction of plant proteins for two dimensional electrophoresis Electrophoresis 9 712 718 1988 Englard S and Seifter S Precipitation techniques Methods Enzymol 182 285 300 1990 Cremer F and Van de Walle C Method for extraction of proteins from green plant tissues for two dimensional polyacrylamide gel electrophoresis Anal Biochem 147 22 26 1985 Guy G R et al Analysis of cellular phosphoproteins by two dimensional gel electrophoresis applications for cell signaling in normal and cancer cells Electrophoresis 15 417 440 1994 Meyer Y et al Preparation by two dimensional electrophoresis of proteins for antibody production antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplasts Electrophoresis 9 704 712 1988 Halloway P and Arundel P High resolution two dimensional electrophoresis of plant proteins Anal Biochem 172 8 15 1988 Flengsrud R
247. rylamide gels for the second dimension separation Sample can be loaded using rehydration loading cup loading or paper bridge loading Each of these is described in more detail later in this chapter Figure 45 provides guidelines for selecting the appropriate mode of sample application 80 6429 60 AD 91 Multiphor II Electrophoresis System pH gradient Analytical Preparative 3 5 4 5 3 0 5 6 NL 4 0 7 0 3 0 7 0 NL 5 3 6 5 3 0 10 0 3 0 10 0 NL 3 0 11 0 NL 6 2 7 5 6 0 9 0 6 0 11 0 7 0 11 0 NL Cup loading Fig 45 Guidelines for selecting the appropriate mode of sample application in the Multiphor II Electrophoresis System For cup loading sample is pipetted into sample cups precisely positioned on the surface of the Immobiline DryStrip gels Up to 100 ul per strip can be applied through sample cups and up to 850 ul with paper bridge loading 74 4 1 1 Immobiline DryStrip gel rehydration IPGbox IPGbox allows up to 12 strips up to 24 cm long to be rehydrated independently and simultaneously Samples can be loaded during rehydration by including them in the rehydration buffer rehydration loading Alternatively samples can be applied to rehydrated strips via sample cups or paper bridge loading For protocol see section 2 7 page 57 Table 35 Rehydration solution volume per Immobiline DryStrip gel Multiphor II protocol Immobiline DryStrip gel length cm Total volume
248. rylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G 250 and R 250 Electrophoresis 9 255 262 1988 Yan J X et al A modified silver staining protocol for visualization of proteins compatible with matrix assisted laser desorption ionization and electrospray ionization mass spectrometry Electrophoresis 21 3666 3672 2000 Fernandez Patron C et al Understanding the mechanism of the zinc ion stains of biomacromolecules in electrophoresis gels generalization of the reverse staining technique Electrophoresis 19 2398 2406 1998 Mackintosh J A et al A fluorescent natural product for ultra sensitive detection of proteins in one dimensional and two dimensional gel electrophoresis Proteomics 3 2273 2288 2003 Steinberg T H et al Applications of SYPRO Orange and SYPRO Red protein gel stains Anal Biochem 239 238 245 1996 80 6429 60 AD 147 88 89 90 9 92 93 94 9 96 9 98 j in 5N Steinberg T H et al Fluorescence detection of proteins in sodium dodecylsulphate polyacrylamide gels using environmental benign nonfixative saline solution Electrophoresis 21 497 508 2000 Patton W Detecting proteins in polyacrylamide gels and on electroblot membranes in Proteomics from protein sequence to function Pennington S R and Dunn M J eds Bios Scientific Publishers Ltd pp 65 86 2001 Ya
249. s continued Contaminant Reason for removal Removal techniques Insoluble material Insoluble material in the sample can clog Samples should always be clarified by gel pores and result in poor focusing centrifugation prior to application in Insoluble material is particularly problematic first dimension IEF when the sample is applied using sample cups as it can prevent protein entry into the IPG strip Even relatively low concentrations of salts lt 5 mM can slow down separation prevent sharp focusing or cause disturbances that result in a poor quality 2 D result Low molecular weight ionic impurities can originate either as endogenous components of the sample source or as salts and buffers introduced during preparation of the sample In either case the ability of a sample to be effectively separated by first dimension IEF and the subsequent quality of the 2 D electrophoresis result can often be improved by dialyzing the sample prior to application Mini Dialysis Kit is well suited for this application because the capacity of the dialysis tubes 10 250 ul or 200 2000 ul corresponds to typical volume ranges for 2 D samples and because sample losses from the procedure are negligible 1 5 1 Desalting samples using Mini Dialysis Kit Mini Dialysis Kit is designed for the dialysis of small sample volumes with minimal handling and sample loss offering a simple solution to the handling problems of low volume dialysis and reducing the pron
250. s reposition the cups remove the cover fluid with a pipette and check for leakage again An optional check for leakage is to add 0 01 bromophenol blue dye solution to the cups If the dye leaks out of a cup it must be corrected Important the leaked detection dye must be removed from the sample cup before loading the sample Low current This is normal for Immobiline DryStrip gel s which have very low conductivity An Immobiline DryStrip gel run usually starts at 50 100 pA strip and drops during the run to below 10 pA strip EPS 3501 XL Power Supply cannot detect the low pA range current and shuts off Because the EPS 3501 XL Power Supply can operate under very low currents it is recommended for use with Immobiline DryStrip Kit and Immobiline DryStrip gels Make sure the low current shut off has been bypassed see 3501 XL Power Supply instructions IPG runs may start in a current range that is not detectable by the EPS 3501 XL Power Supply IPG Buffer omitted from rehydration solution Always include IPG Buffer or Pharmalyte in the rehydration solution No current at start of run No electrod of electrical e contact or lack continuity Check that all Multiphor II contacts are in place Make sure the metal band within the electrode contacts the metal band along the side of the Immobiline DryStrip tray Note that the metal band within the electrode is only on the end marked with th
251. s 44 2 2 2 CHOOSING the PH Gradient oa ceccceeecccccccssssssssssssssssssssesssesesseceeeeeesesesssssssssssussnussssseseseeeeeeceeeeeessssensssssass 45 2 2 3 EMOOSIAGHON IPG BUE ias sssini a a iaa 46 2 2 4 Estimating the pl of proteins 46 2 3 IEF using Ettan IPGphor 3 Isoelectric Focusing System and Accessories s 47 2 3 2 Ettan IPGphor 3 MAMIO aisisiiiisiserisisrinisnisaiaisarinaniniinns 48 23 PODO rnane e gee ee ates E E EAS 51 2 3 4 Ettan IPGphor 3 Strip Holders oee ccccecssssssssssssssssssessssessssececscceeccesssssssssnsnsnnusssessesecseceeeeeeeesessasssssaass 51 23 5 General COULIOMS ssie n i iii inah 51 ZA Selecting sample application MELO sic cscccsscsiccssrszsrsssoccasassasesesvasacuceacates ansdsacacsbancasnteceuivenadaatecd eivacsecistee 52 241 REMY ARO OM IOC AIAG eesin anean OR RRE 52 242 NY SECOT MMIC Gl iranneseeoean ee iaa e E E AOR 52 245 Pa perbridge l ddiNgi serieou iE AORE 52 2 5 Recommended sample LOGS sssssssssssssssssesssesessesceececescssssssssssnuunusssssceseeeeceeecessessssssnsnsunsussssssceeeseeeeeeetes 53 2 6 Immobiline DryStrip gel rehydration SOlUtIONS ccccssssssssssssssessssessesssescceseessessssssssssssuuunessssseseeseeseee 53 2 6 1 Components of rehydration SOLUTION ceeeeesscsessessssccccecesssssssssnsnnunussssesesseseeeeeeeeeesesssssssnsass 54 2 6 2 Using DeStreak Rehydration Solution ceeessscssssssscsccececesssssssssssnusessesseseeeceeseeesssssssesseass 55 2 6 3 Prep
252. s no discrete application point this method eliminates the formation of precipitates at the application point that may occur when loading using sample cups Also the method is technically simpler than the others avoiding problems of leakage that can occur when using sample cups 2 4 2 Use of Manifold There are cases when it may be preferable to load the sample following rehydration immediately prior to IEF For example if proteolysis or other protein modifications are a concern overnight rehydration with sample may not be desirable The Manifold see section 2 7 provides a convenient means to load samples under such circumstances Cup loading using the Manifold is recommended for sample volumes up to 150 ul and a maximum protein concentration of 150 ug protein 150 pl sample solution 150 pl is the volume of the cup Larger sample loads can lead to increased protein precipitation at the point of application Anodic cup loading has been found to improve protein 2 D spot patterns with basic Immobiline DryStrip gels pH 6 9 pH 6 11 and pH 7 11 NL Under conditions where substantial water transport electroendosmosis accompanies focusing such as with protein loads in excess of 1 mg the face up mode frequently yields better resolution See section 2 3 4 for a more detailed discussion of the face up mode 2 4 3 Paper bridge loading Paper bridge loading is ideal for very large sample volumes and preparative electrophoresis and is particularly
253. s used for SDS gels can be applied to second dimension gels The following features are desirable e High sensitivity e Wide linear range for quantitation e Compatibility with mass spectrometry e Low toxicity and environmentally friendly However because none of the existing techniques can meet all these requirements a 2 D electrophoresis laboratory may need to have more than one of the following methods in its repertoire Autoradiography and fluorography are the most sensitive detection methods down to 200 fg of protein To employ these techniques the sample must contain protein radiolabeled in vivo using either 35S 1 C 3H or in the case of phosphoproteins 3P or 3P For autoradiographic detection the gel is simply dried and exposed to X ray film or for quicker results and superior dynamic range of quantitation to a storage phosphor screen Fluorography is a technique that provides extra sensitivity by impregnating the gel in a scintillant such as PPO 2 4 diphenyloxazole prior to drying Silver staining is a sensitive non radioactive method below 1 ng Silver staining is a complex multi step process utilizing numerous reagents for which quality is critical It is therefore often advantageous to purchase these reagents in the form of a dedicated kit in which the reagents are quality ensured specifically for the silver staining application PlusOne Silver Staining Kit Protein combines high sensitivity with ease of use By omit
254. sample volume to ensure the resin remains in suspension during the incubation period Disposable 15 ml centrifuge tubes are recommended 2 Add 750 ul of the suspended slurry to the tube containing the sample It is essential that the gel slurry is homogenous uniform suspension prior to pipetting When dispensing the resin it may be easier to pipette if the narrow end of the tip is cut off This would normally be performed with scissors prior to use 3 Mix the gel sample mixture on a rotary shaker for a minimum of 30 min at room temperature Mixing speed should be sufficient to keep the gel sample in suspension Rapid rotary shaking is required to ensure that the resin remains in suspension Speeds in the region of 250 rpm are recommended 4 Snap off the base tip of the microspin column Place each column into a microcentrifuge tube supplied 5 Atthe end of the incubation period make sure that the resin is in suspension and carefully pipette the gel sample mixture into the upper chamber of the microspin column which is sitting in a microcentrifuge tube Ensure that all liquid is removed from the tube If drops of liquid are splashed around the incubation tube briefly centrifuge the incubation tube and contents 1000 rpm 2 min to collect the resin and liquid prior to transfer to the microspin column 6 Centrifuge at approximately 6500 x g for 5 min Note The resin should appear dry and powdery following centrifugation 7 Di
255. scard the upper chamber containing the gel and collect the filtrate Note The approximate volume of the filtrate will be 500 yl 8 The sample is now ready to be used immediately for further processing or stored for later use ce If proteins are to be analyzed by 2 D gel analysis concentration and desalting will be required Protocol for acetone precipitation not required if using 2 D Clean Up Kit 1 Place an aliquot of acetone at 20 C at least 15 min prior to use Approximately 2 ml of acetone is required for each sample Ensure that the tube is made of an acetone compatible material 2 Accurately measure the volume of each sample Divide each sample volume into two microcentrifuge tubes Add 4 volumes of ice cold acetone to the sample volume in each tube 36 80 6429 60 AD 1 5 ml hinged microcentrifuge tubes are recommended Small pellets are easily visualized using this type of tube 3 Allow proteins to precipitate at 20 C for at least 2 h 4 Pellet proteins by centrifugation at approximately 13 000 x g 13 000 rpm in a small bench microcentrifuge for 10 min at 2 8 C Place the tubes into the microcentrifuge in known orientation Place the hinged lid outward as this assists with detection of small protein pellets 5 Decant off the acetone but do not disturb the pellet A clean tissue can be used to carefully remove any spots of acetone away from the pellet 6 Allow pellets to air dry typically 5 10 min at ro
256. sccticscsticscstssesctsdiravttiaunssicleadahasaats a a E A 20 DDL GOMtle YSIS MELA OMS icc csssseecssskiscecceseccnsscccsssdesoesssaniasshvenegssesseasidesonnsasnecsbcateccianonsaseesiassasadacenendassctcnd 20 1 1 2 More Vigorous lysis Method S si wel 1 1 3 Processing small tissue or cell samples using Sample Grinding Kit wn cccccccccccee 21 1 1 4 Preparing samples from difficult protein SOUPCES wecceececcccccsssssssssssssssssssssescessssseesseeeeee 22 1 2 Protecting AGAINSt proteolysis o eeeccccsssssssssssssssesssesessecceeceseesessssssssssssmsssssesesseseeeeeeeseessssssssssussuuueessees 23 1 2 1 Protease inhibition using Protease Inhibitor MIX i eccccccssssccsccscsesssssssssssnusssscessssseceeceeeee 24 13 Fractionation OF PROTEIN YS GCE c sccscscsciscessacscvslactsassasasbececdoaunagsopscocsossssteysscusayesastevensctbgeesseenaacotspcntacet ceteiet 24 1 4 Precipitation PRO CSA ULES 5 ccase cialscaccasdeacescnscavscusnasassassshaonsiestacasvsvascb tod asasteavaseatesasctspnscsadasscbebbphesechebinsstgassa dens 25 1 4 1 Cleaning up samples using 2 D Clean Up Kit 26 1 4 2 RESUSPENSION Of Pellet ne sssssscccccececeessssssssunsnssssesseseeseeeeeceeesssssssssssssuussssesseseeeseeeeeeeeees 29 1 5 Other methods for removing CONLAMINANES wn ceccccccscssssccccccesssssssssssssnsssssesesessecceeeeeessesssssssssssssuuueensees 30 1 5 1 Desalting samples using Mini Dialysis Kit sssssessssccccccccecssssssssssnsnussessessssseeeeeeeeees 32 1 5 2 Removing
257. separation methods a drawback when complex protein lysates are run on 2 D gels without prefractionation is that the resulting 2 D gel is crowded with spots making interpretation of results difficult Typically a 2 D gel can yield anywhere between 1000 and 4000 spots under favorable conditions but the presence of many of the most interesting proteins particularly low abundance proteins can be masked Where high protein loads are employed such as with preparative 2 D gels higher protein amount loaded onto the gel results in 2 D patterns having poorer resolution with spots of very abundant proteins overlaying the spots of less abundant proteins If the loads are increased even more abundant proteins become predominant and the separation is poor Thus the greatest challenge in protein discovery and analysis of important proteins is the right sample preparation strategy for 2 D electrophoresis Strategies for prefractionation of samples for 2 D electrophoresis appear to be the most promising approach for increasing the number of protein components that can be visualized in complex proteomes such as mammalian cells tissues and physiological fluids In addition removal of contaminants is part of the strategy For this purpose GE Healthcare provides the Albumin and IgG Removal Kit which includes an affinity gel to selectively remove albumin and IgG contaminants in human serum 2 D maps The use of Albumin and IgG Removal Kit to improve 2 D electrophores
258. sing IEF separates proteins according to their isoelectric points pl the second dimension step sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE separates proteins according to their molecular weights M relative molecular mass Each spot on the resulting two dimensional gel potentially corresponds to a single protein species in the sample Thousands of different proteins can thus be separated and information such as the protein pl the apparent molecular weight and the amount of each protein can be obtained Two dimensional electrophoresis was first introduced by O Farrell 1 in 1975 In the original technique the first dimension separation was performed in carrier ampholyte containing polyacrylamide gels cast in narrow tubes See section 2 1 on page 43 for more details The power of 2 D electrophoresis as a biochemical separation technique has been recognized virtually since its introduction Its application however has become significant as a result of a number of developments e The introduction of immobilized pH gradients and Immobiline reagents 2 brought superior resolution and reproducibility to first dimension IEF Based on this concept G rg and colleagues 3 4 developed the currently employed 2 D technique where carrier ampholyte generated pH gradients have been replaced with immobilized pH gradients and tube gels replaced with gels supported by a plastic backing A more detailed discussion of the mer
259. solution 30 acrylamide 0 8 N N methylenebisacrylamide 1 Final concentration Amount Acrylamide FW 71 08 30 300g N N methylenebisacrylamide FW 154 17 0 8 8g Double distilled water to1l Filter solution through a 0 45 um filter Store at 4 C in the dark H 4x resolving gel buffer solution 1 5 M Tris base pH 8 8 1 Final concentration Amount Tris base FW 121 1 1 5 M 181 7 g Double distilled water 750 ml HCl a adjust to pH 8 8 Double distilled water to1l Filter solution through a 0 45 um filter Store at 4 C l Bromophenol blue stock solution Final concentration Amount Bromophenol blue 1 100 mg Tris base 50 mM 60 mg Double distilled water to 10 ml J 10 SDS solution 10 SDS 50 ml Final concentration Amount SDS FW 288 38 10 w v 5 0g Double distilled water to 50 ml Filter solution through a 0 45 um filter Store at room temperature 80 6429 60 AD 131 K 10 ammonium persulfate solution 10 ammonium persulfate 10 ml and 1 ml Final concentration Amount for 10 ml Amount for 1 ml Ammonium persulfate FW 228 20 10 w v 1 0g 0 1g Double distilled water to 10 ml tolml Fresh ammonium persulfate crackles when water is added If it does not replace it with fresh stock Prepare just prior to use L Gel storage solution 375 mM Tris HCI 0 1 SDS 1 Final concentration Amount 4x Resolving gel buffer see solution H above 1x 250 ml
260. ss Er Perform a literature search to determine if others have already worked out a sample preparation strategy g Discussion groups such as the one at www gehealthcare com can also be helpful The cells or tissue should be disrupted in such a way as to minimize proteolysis and other types of protein degradation Cell disruption should be performed at as low a temperature as possible and with a minimum of heat generation Cell disruption should ideally be carried out directly into a strongly denaturing solution containing protease inhibitors Preserve sample quality by preparing the sample just prior to IEF or storing samples in aliquots at 40 C or below Do not expose samples to repeated freezing and thawing Remove all particulate material by ultracentrifugation Solid particles and lipids must be removed because they will block the pores in the electrophoresis gel To avoid modification of proteins never heat a sample after adding urea If the sample contains urea the solution temperature must not exceed 37 C Elevated temperatures cause urea to hydrolyze to isocyanate which modifies proteins by carbamylation resulting in artifactual charge trains E This chapter describes methods of sample preparation for 2 D electrophoresis using precast Immobiline DryStrip gels available from GE Healthcare Optimal protein loads for Immobiline DryStrip gels are discussed in section 2 5 For more information on using Immobiline DryStrip g
261. ssignment of sequence information A large and growing application of 2 D electrophoresis is within the field of proteomics 6 7 The analysis involves the systematic separation identification and quantitation of many proteins simultaneously from a single sample Two dimensional electrophoresis is used in this field due to its unparalleled ability to separate thousands of proteins simultaneously The technique is also unique in its ability to detect post and co translational modifications which cannot be predicted from the genome sequence Applications of 2 D electrophoresis include proteome analysis cell differentiation detection of disease markers therapy monitoring drug discovery cancer research purity checks and microscale protein purification This handbook describes methods for 2 D electrophoresis using precast IPG strips Immobiline DryStrip gels available from GE Healthcare Symbols used in this handbook Er This symbol indicates general advice that can improve procedures or provide recommendations for action under specific situations W This symbol denotes advice that should be regarded as mandatory and gives a warning when special care should be taken L This symbol highlights troubleshooting advice to help analyze and resolve difficulties that may occur chemicals buffers and equipment experimental protocol 8 80 6429 60 AD Table 1 Equipment choices for 2 D electrophoresis A First and second dimension electrop
262. sssescesceccccsssssssssssssnunutsssesssseeseceeececcesesssssssssnnasususssscseeseseeeeeeeees 95 4 1 5 IEF guidelines for Multiphor Il Electrophoresis System ac ccccccccssssssssssssssssssssessecsssseseseeeesees 96 ALG Prot col xa mpl Sisian 96 4 1 7 Running a Multiphor Il Protocol nec cccccccccsceesssesee 97 4 1 8 Preservation of focused Immobiline DryStrip gels 99 AES TROUDISSMO OEING 22eessdscesssdececcedectedsckssssesdtastacactedactaxdisecitchs Piseasesstiaesaassscleuste EA 100 4 2 Second Dimension SDS PAGE using Multiphor II Electrophoresis SYSteM cccccscsssssse 101 421 ExcelGel OSPR OM sosina N a a 101 4 2 2 Applying equilibrated Immobiline DryStrip Gels esscsssssscccccecccssssssssssssssnessesesesees 102 4 2 3 Electrophoresis Conditions eeeesssssssscssssscccccccceessssssssnsnuuunmssscssesesseseeeeeesessssssssssanusnunussecsesees 103 AOA THOU ISSHOOEING essene ea an ii i N 104 5 Visualizing and evaluating results se sessssssssesrsssressssssssssrresssesssesssneensreressresssesesssseneesntesneneesneseness 105 5 0 Visualizing results labeling and Staining wcceecccccsccssscssssscsssesssssssssssnuusssssssssesececeeceeesesssssssssssnuenseesees 105 5 0 1 Automating processing and preserving the gel u cccccssssssssssssssssesccsessesscceessssesssssssnsssneeee 106 NE NG crea T A A E AA 107 5 2 EVO OG OSUNS rnea aO AREER ONARE 107 55 Standardiang SSCS p esrsinerne e E EEA AATE E 108 5 4 Further analysis of protein SPots ssssss
263. sssssssssssccssssscscccccecscessssssssssnumussssesseseeeeeeeeeessssssssssssnnuensseseess 108 54L PICKING Prote SOLS enep EE N RE RNE 108 5 4 2 Digesting proteins and spotting onto MALDI TOF MS slideS i eeccccscsscscccscssessssssssnsnneeee 108 5 4 3 MALDI TOF mass spectrometry uu ceeeccssssessssssetssesssssescessnsesecsssseeceesnnscsesssnsseessnseseessneesseesneteeeeseneses 108 6 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE sssesesssssssssssseseseesssessresesreeesrereseee 111 6 0 Ove Vie Wssenenninennnianennsnnaniusni annei sa LII Gil CyDy amp DIGE FUON AOS scccssiesec stccssvstusessstassscnsteasheivcscosanchessccessussassyehictitoenstcnantvabasesdeceusdsatarwatast deiabeaserconstenten 113 6 1 1 CyDy DIGE Fluor minal dye Siersema 113 6 1 2 Minimal labeling of protein with CyDye DIGE Fluor minimal dy S s1 111211 1112 114 6 2 CyDye DIGE Fluor labeling kits with saturation dyes for labeling scarce samples and preparative GEIS cccscssssccccccccsessessssssssnsnsescesesseseeeeeeecseseesssssssenneeee 114 6 3 Ettan DIGE system WOrkflOW cssssessssssesscsssssecccccecssssssssssnsnunsmssssssesseseeseesessesssssssssssnnsmuscsseseseeeeeseeseessses 116 6 3 1 Experimental design for Ettan DIGE system applications cece 117 6 3 2 Sample preparation for Ettan DIGE system applications ccceeccccccsssssssssssssseeeseseee 119 6 3 3 Sample labeling with minimal dyes for Ettan DIGE system applications 120 6 3 4 Two dimensiona
264. stem Volume ml miniVE and SE 260 10 x 10 5 cm plates 1 mm thick spacers 10 1 5 mm thick spacers 15 SE 600 Ruby 18 x 16 cm plates 2 cm wide x 1 mm thick spacers 30 2 cm wide x 1 5 mm thick spacers 40 1 cm wide x 1 mm thick spacers 30 1 cm wide x 1 5 mm thick spacers 45 80 6429 60 AD 85 Table 30 Single percentage gel recipes for miniVE SE 260 and SE 600 Ruby systems Final gel concentration 5 7 5 10 12 5 15 Monomer solution solution G 16 7 ml 25 ml 33 3 ml 41 7 ml 50 ml 4x resolving gel buffer solution H 25 ml 25 ml 25 ml 25 ml 25 ml 10 SDS solution J 1ml 1ml 1ml 1ml 1ml Double distilled water 56 8 ml 48 5 ml 40 2 ml 31 8 ml 23 5 ml 10 ammonium persulfatet solution K 500 ul 500 ul 500 ul 500 ul 500 ul TEMEDt 33 ul 33 ul 33 ul 33 ul 33 ul Total volume 100 ml 100 ml 100 ml 100 ml 100 ml Preparation of stock solutions is described in appendix I solutions G H J and K t Ammonium persulfate and TEMED are added immediately prior to casting the gel Table 31 Recipes for gradient gels for miniVE SE 260 and SE 600 Ruby systems Light solution Final concentration 5 7 5 10 12 5 15 Monomer solution solution G 8 4 ml 12 5 ml 16 5 ml 21 0 ml 25 ml 4x resolving gel buffer solution H 12 5 ml 12 5 ml 12 5 ml 12 5 ml 12 5 ml 10 SDS solution J 500 ul 500 ul 500 ul 500 ul 500 ul Double distilled water 28 5 ml 24 5 ml 20 0 ml 16 0 ml 12 0 m
265. stic wrap Alternatively the DryStrip gels can be stored in screw cap tubes The 7 cm strips fit in disposable 15 ml conical tubes 11 13 and 18 cm strips fit in 25 x 200 mm screw cap culture tubes and 18 and 24 cm strips fit in Equilibration Tubes see ordering information The equilibration process is discussed in chapter 3 80 6429 60 AD 67 2 9 Troubleshooting L Table 20 lists possible problems that might be encountered during IEF and how to solve them and Table 21 lists problems and solutions when using Ettan Manifold Table 20 Troubleshooting first dimension IEF Ettan IPGphor 3 Isoelectric Focusing System Symptom Possible solutions Problems indicated by LCD messages Lid open step 1 close to continue The safety lid is not properly closed When the safety lid is open the system has an automatic voltage cutoff safety feature In order for the protocol to proceed the safety lid must be closed Locked screen in edit mode Turn off the mains power switch to reset the instrument Blank display f no electrical components are functioning e g HV lamp does not light and the cooling fans are motionless check the fuses in the mains power module Diagnostic program indicates component failure Note the component that failed and press the START key to continue hrough the diagnostic program Call your local GE Healthcare sales office for further information on how to remedy the failure
266. sume a normal voltage step program This allows the ions in the sample to move to the ends of the Immobiline DryStrip gel continues on following page 126 80 6429 60 AD Table 44 Troubleshooting 2 D results continued Symptom Possible cause Remedy lonic detergent in sample If the ionic detergent SDS is used in sample preparation the final concentration must not exceed 0 25 after dilution into the rehydration solution Additionally the concentration of the nonionic detergent present must be at least eight times higher than the concentration of any ionic detergent to ensure complete removal of SDS from the protein Horizontal stripes across gel Prominent vertical streak at the point of sample application when loading Immobiline DryStrip gels and sample cups Impurities in agarose overlay or equilibration solution Flatbed gel format Sample aggregation or precipitation Prepare fresh agarose overlay and equilibration solution Dilute the sample and apply as a larger volume Program a low initial voltage and increase voltage gradually Impurities in sample Modify sample preparation See section 1 4 Impurities in rehydration solution components Use only high quality reagents Deionize urea solutions Bubble between Immobiline DryStrip gel and top surface of second dimension gel Ensure that no bubbles are trapped between the Immobiline DryStrip gel an
267. suspension 2 Add 10 ul of Nuclease Mix per 1 ml reaction mix Vortex briefly and incubate at room temperature for 30 45 min 1 5 3 Simultaneous DNA RNA and protein isolation from undivided scarce samples The illustra triplePrep Kit is designed for the rapid extraction of genomic DNA total RNA and total denatured protein from a single undivided sample This kit can be used for removing undesirable nucleic acids For more information on protocol see product booklet 28 9425 44 1 5 4 Using Albumin and IgG Removal Kit to improve 2 D electrophoresis of human serum Proteins in serum and other biological fluids are difficult to resolve by 2 D electrophoresis largely due to the abundance of serum albumin and IgG In human serum albumin constitutes 50 70 of the total protein and IgG constitutes 10 25 The presence of these proteins obscures other proteins in the gel and limits the amounts of proteins in the serum that can be resolved by 2 D electrophoresis In addition these proteins have wide pl and molecular weight ranges that further reduce resolution and mask some low abundance proteins Albumin and IgG Removal Kit improves resolution of low abundance proteins and increases the number of spots in the treated sample The kit includes an affinity gel that selectively binds human albumin and IgG and enhances the visibility of low abundance proteins Albumin and IgG Removal Kit improves on the currently available Cibacron Blue dye based technolo
268. t co precipitant copper solution color reagent A color reagent B bovine serum albumin BSA standard solution Required but not provided Microcentrifuge microcentrifuge tubes 2 ml vortex mixer visible light spectrophotometer spectrophotometer cells Preliminary notes Prior to starting procedure prepare working color reagent by mixing 100 parts of color reagent A with one part of color reagent B Each individual assay requires 1 ml working color reagent 1 Prepare standard curve 0 50 ug using the 2 mg ml BSA standard solution 2 Prepare microcentrifuge tubes in duplicate containing 1 50 ul of the sample to be assayed The useful range of the assay is 0 5 50 yg 80 6429 60 AD 39 9 10 E NZ Add 500 ul precipitant to each microcentrifuge tube including tubes containing the BSA standard Vortex and incubate 2 3 min at room temperature Add 500 ul co precipitant Mix briefly Centrifuge at least 10 000 x g for 5 min Remove supernatant Centrifuge briefly to bring remaining supernatant to bottom of tube Remove remaining supernatant with micropipette ae Proceed rapidly to avoid resuspension or dispersion of pellet There should be no visible liquid remaining Add 100 ul copper solution and 400 ul distilled or deionized water to each tube Vortex to dissolve the precipitated protein cer Ensure that the pellet is completely resuspended by vortexing thoroughly Add 1 ml working color reagent to each tube
269. t Immobiline DryStrip gels eliminate the need to handle toxic acrylamide monomers In addition preparation time and effort are significantly reduced and reproducibility of the pH gradient is ensured Additional advantages of Immobiline DryStrip gels include e Immobilized pH gradients and precise lengths ensure high reproducibility and reliable gel to gel comparisons e To simplify gel use and record keeping each strip is labeled with the pH interval and a unique identifier and won wn bar coded for use with a reader A or sign indicates the anodic or cathodic side of the strip respectively 80 6429 60 AD 43 e Immobiline DryStrip gels are compatible with Ettan DIGE system the most powerful approach for comparative analysis of relative protein abundance using 2 D electrophoresis see chapter 6 Figure 18 illustrates the pH intervals of Immobiline DryStrip gels The most recent additions to the product line comprise two sets of pH interval a broad range gel pH 3 11 NL and four medium range gels covering shorter pH intervals pH 3 5 6 NL 5 3 6 5 6 2 7 5 and 7 11 NL These four medium range gradients overlap each other in an optimized way allowing outstanding coverage of all proteins in the pH 3 to 11 range with improved separation in the extreme basic pH region of the IPG strips Narrow range Immobiline DryStrip gels covering just one pH unit are a valuable complement to the newer strips in many experimental situations
270. t be determined empirically for each sample type Focusing after sample cup application frequently requires fewer Vhr than in gel sample rehydration loading methods particularly on basic pH range strips Incorrect cup position Correct cup position j Fig 29 Manifold placement on Ettan IPGphor 3 5 C C Fig 31 Sample cup positioning details SS Note Cups must not straddle the centering protrusions on the 3 9 R R Q Q bottom of the channels HI ee 24 cm b qp 4p q p i L aem ie 13 cm 11 cm 4 Fig 32 Correct placement of paper wicks siege gBigeigialis LY Fig 30 Placement of IPG strips in Manifold channels Note If cathodic cup loading is going to be used the strips should be placed such that the anodic end of the strips is 3 4 cm Fig 33 Placement of electrode on paper wicks Cams are in the beyond the etched placement mark open position 62 80 6429 60 AD 2 8 Isoelectric focusing guidelines Ettan IPGphor 3 System IEF using the Ettan IPGphor 3 Isoelectric Focusing System is conducted at very high voltages up to 10 000 V depending on the length of the DryStrip use
271. tan 30 SE 751 84 Uppsala Sweden and any money paid for the material will be refunded DeCyder This release of DeCyder software is provided by GE Healthcare to the customer under a nonexclusive license and is subject to terms and conditions set out in the 2 D Differential Gel Electrophoresis Technology Access Agreement Customer has no rights to copy or duplicate or amend the Software without the prior written approval of GE Healthcare Deep Purple Total Protein Stain is exclusively licensed to GE Healthcare from Fluorotechnics Pty Ltd Deep Purple Total Protein Stain may only be used for applications in life science research Deep Purple is covered under a granted patent in New Zealand entitled Fluorescent Compounds patent number 522291 and equivalent patents and patent applications in other countries DIGE Gel and DIGE Buffer Kit The buffer system in this gel and buffer kit is covered by patent application WO9616724 granted in US EP and JP Ettan CAF MALDI Sequencing Kits are protected by patents owned by Procter amp Gamble Company and exclusively licensed to GE Healthcare Bio Sciences AB and by joint patents issued to both companies The purchase of Ettan CAF MALDI Sequencing Kits includes a limited license to use the technology for internal research and development but not for any commercial purposes No right to perform or offer commercial services or products of any kind using the Sequencing Kits is hereby granted A license to us
272. tardialdehyde from the sensitizer and formaldehyde from the silver nitrate solution as well as omitting the preservative step the method becomes compatible with mass spectrometry analysis although sensitivity is reduced If glutardialdehyde and formaldehyde are to be used add them just before staining The volume of the formaldehyde in the developing solution can be varied from 100 ul up to 250 ul depending on the amount of protein and the number of spots since formaldehyde is consumed in the developing reaction by proteins Add the formaldehyde directly before use Approximate time this step may be visually monitored The gels should be transferred to stop solution when the spots have reached the desired intensity and before the staining background becomes too dark For gels cast on plastic supports increase the amount of glycerol to 25 ml Short and long term storage of gels is possible in 10 ethanol rather than glycerol if gels are not being dried down Glycerol is necessary only if planning to dry down gels Storage in ethanol allows the gels to be compatible with spot picking mass spectrometry 80 6429 60 AD 133 134 80 6429 60 AD Appendix III Colloidal Coomassie staining procedure This method has been modified from Neuhoff et al 83 5 Coomassie Blue G 250 stock 5 Coomassie Blue G 250 10 ml Amount Coomassie Blue G 250 0 5g Double distilled water to 10 ml Stir for a few minutes to disperse the
273. te volume of SDS equilibration buffer solution see appendix solution E then measure into two equal volumes Add DTT to one portion 100 mg per 10 ml and iodoacetamide to the other 250 mg per 10 ml Equilibration 1 Add the appropriate volume of SDS equilibration buffer DTT to each strip Cap or seal the tubes with flexible paraffin film and place them on their sides on a rocker for the equilibration process Equilibrate for 15 min 2 Pour off buffer from above step and add the appropriate volume of SDS equilibration buffer iodoacetamide to each strip Again cap or seal the tubes with flexible paraffin film and place them on their sides on a rocker for the equilibration process Equilibrate for an additional 15 min EF Be consistent with the timing of the equilibration steps Table 22 Suggested containers and volumes of equilibration solution Strip length cm Container Equilibration solution ml 7 Disposable 15 ml conical tubes 2 5 5 11 25 x 200 mm screw cap culture tubes 5 10 13 25 x 200 mm screw cap culture tubes 5 10 18 25 x 200 mm screw cap culture tubes Equilibration 10 15 tubes available from GE Healthcare or Petri dish 24 Equilibration tubes available from GE Healthcare or Petri dish 10 15 Er The subsequent steps of gel assembly preparation of electrophoresis unit insertion of the gel into the precast gel cassette and melting of the sealing solution can be performed as the Immobiline DryStrip g
274. th blanks 2 When all six slots are occupied adjust the buffer level with distilled water so that the level of the diluted anode buffer is at the LBC start fill line marked on the unit 3 Seat the upper buffer chamber over the gels Fig 43 oe Lubricate the gasket and cassette with cathodic buffer e g SDS running buffer to assist in assembly 4 Fill the UBC with 1 2 of 2x buffer if not already done in the protocol in section 3 3 1 5 Using a small funnel quickly fill the narrow space between the upper and lower buffer chambers with anode buffer or distilled water to the same level as in the upper buffer chamber It is important that the anode and cathode buffers are filled to the same height in the Ettan DALTsix buffer chambers 6 Attach and close the lid Connect the power leads to the power supply Fig 42 Inserting the cassettes into the cassette carrier Fig 43 Seating the upper buffer chamber 78 80 6429 60 AD Protocol Inserting gels into Ettan DALTtwelve When the electrophoresis buffer has reached the desired temperature insert the loaded gel cassettes with the Immobiline DryStrip gels in place Gel Cassettes and Blank Cassette Inserts slide much more easily into the unit if they are wet Distilled or deionized water from a squirt bottle can be used to wet the cassettes and Blank Cassette Inserts as they are being loaded into the unit 1 Load the unit from back to front Fig 44 2 Fit Blank Cass
275. the forceps and lift the strip out of the tray Position the Immobiline DryStrip gel in the Immobiline DryStrip aligner Immediately transfer the rehydrated Immobiline DryStrip gels gel side up to adjacent grooves of the aligner in the Immobiline DryStrip tray Fig 48 Place the strips with the acidic ends at the top of the tray near the red electrode anode The other ends should be at the bottom of the tray near the black electrode cathode Align the Immobiline DryStrip gels so the anodic gel edges are lined up Attach the electrode strips Place the moistened electrode strips laterally across the cathodic and anodic ends of the aligned Immobiline DryStrip gels The electrode strips must at least be in partial contact with the gel surface of each Immobiline DryStrip gel 80 6429 60 AD 93 4 Position the electrodes Each electrode has a side marked red anode or black cathode Align each electrode over an electrode strip ensuring the marked side corresponds to the side of the tray giving electrical contact When the electrodes are properly aligned press them down to contact the electrode strips Check that the Immobiline DryStrip gels are still aligned in their grooves Fig 49 5 Overlay the Immobiline DryStrip gel with Immobiline DryStrip Cover Fluid Overlay each Immobiline DryStrip gel with 3 ml of Immobiline DryStrip Cover Fluid to minimize evaporation and urea crystallization Fig 48 Positioning Immobiline DryStrip gels i
276. the pl of proteins The reliability of the first dimension separation is so high that the pl of a protein can be estimated by relating its position on the second dimension gel to its original position on the Immobiline DryStrip Using linear pH gradients increases the accuracy of this estimation Determine the first dimension position by measuring the length of the Immobiline DryStrip gel and the position of the spot on the second dimension gel for gels not attached to backing correct for shrinkage or swelling of the gel during staining Then plot the spot position as a percent of gel length versus pH and read off the pl from the graph of the pH gradient found in Data File 18 1177 60 see additional reading and reference material 46 80 6429 60 AD pH range IPG Buffers 3 5 5 0 Narrow Fig 20 Using IPG strips together with matching IPG Buffer improves the conductivity distribution across the pH gradient during IEF 2 3 IEF using Ettan IPGphor 3 Isoelectric Focusing System and accessories Ettan IPGphor 3 Fig 21 is a fully integrated IEF system optimized to deliver high throughput speed reproducibility and high protein loading capacity as well as optional PC control The large graphical display accommodates multiple up to four lines of text for fast and easy programming Up to10 protocols nine steps each can be saved retrieved and easily edited on the instrument Any number of protocols can be stored on a connected PC runni
277. thoroughly with distilled or deionized water after cleaning vy Handle clean Strip Holders with gloves to avoid contaminating them Strip Holders may be baked boiled or autoclaved DO NOT EXPOSE THEM TO STRONG ACIDS OR BASES INCLUDING ALKALINE DETERGENTS The Strip Holder must be completely dry before use 2 Apply the rehydration solution Pipette the appropriate volume of rehydration solution into each Strip Holder as indicated in Figure 26 Deliver the solution slowly as a stripe of liquid between the two electrodes away from the sample application wells Fig 26 Remove any large bubbles For a typical composition of rehydration solution see appendix solution C If in gel sample rehydration is desired add the appropriate amount of sample to the rehydration solution Recommended loads are shown in Table 16 section 2 5 To ensure complete sample uptake do not exceed the recommended volume of rehydration solution see Table 18 section 2 6 2 3 Position the Immobiline DryStrip gel Remove the protective cover foil from the Immobiline DryStrip gel starting at the acidic end Removal from the acidic end prevents damage to the basic end of the gel which is generally softer Using forceps position the Immobiline DryStrip gel with the gel side down and the anodic end of the strip directed toward the pointed end of the Strip Holder Fig 27 Acidic end first lower the gel onto the solution To help coat the entire strip gently
278. ting glutardialdehyde from the sensitizer and formaldehyde from the silver nitrate solution the method becomes compatible with mass spectrometry analysis 81 although at the expense of sensitivity When staining DALT precast gels with PlusOne Silver Staining Kit Protein a modified staining protocol should be used For details of the modified protocol see appendix II Optimized silver staining of DALT precast gels using PlusOne Silver Staining Kit Protein Coomassie staining although 50 to 100 fold less sensitive than silver staining is a relatively simple method and more quantitative than silver staining Coomassie blue is preferable when relative amounts of protein are to be determined by densitometry Colloidal staining methods are recommended because they show the highest sensitivity down to 100 ng protein spot 82 83 See also appendix III Negative Zinc Imidazole staining has a detection limit of approximately 15 ng protein spot 85 and is compatible with mass spectrometry but is a poor quantitation technique Fluorescent labeling 5 and fluorescent staining 86 provide significant advantages over Coomassie blue or silver staining Fluorescent detection offers increased sensitivity simple robust staining protocols and quantitative reproducibility over a broad dynamic range The method is also compatible with mass spectrometry Deep Purple Total Protein Stain Deep Purple Total Protein Stain from GE Healthcare is a fluoresc
279. ting under the surface of the Immobiline DryStrip Cover Fluid Fig 51 The sample should sink to the bottom of the cup Check for leakage 7 Start IEF Ensure that the electrodes on the tray are connected and place the lid on the Multiphor II unit Connect the leads on the lid to the power supply Ensure that the current check on the EPS 3501 XL Power Supply is switched off Begin IEF ee When sample is applied via sample cups precipitates can form at the application point and the amount of protein that can be loaded is less than if the sample was included in the rehydration solution Protein precipitation and aggregation at the application point can sometimes be avoided by observing the following e The sample should contain urea nonionic detergents and IPG Buffer or carrier ampholytes e Apply the sample in dilute solutions 60 100 ug protein per 100 yl cr For micropreparative applications rehydration loading is recommended Paper bridge loading is recommended if using basic strips see section 4 1 4 2 X Fig 50 Attaching sample cups to the cup bar and pressing them Fig 51 Applying sample into sample cups against Immobiline DryStrip gels 4 1 4 Paper bridge loading Higher sample volumes and protein amounts can be applied with paper bridges that are placed between the anodic or cathodic end of the Immobiline DryStrip gel and the electrode strip A large sample volume requires a large paper pad applied at the opposite end to abso
280. to resuspend and often cannot be fully recovered e The precipitation procedure can itself introduce ions that interfere with first dimension IEF e Precipitation can be time consuming requiring overnight incubation of the sample 2 D Clean Up Kit circumvents these disadvantages by providing a method for selectively precipitating protein for 2 D electrophoresis Protein can be quantitatively precipitated from a variety of sources without interference from detergents chaotropes and other common reagents used to solubilize protein Recovery is generally greater than 90 The procedure does not result in spot gain or loss or changes in spot position relative to untreated samples The precipitated proteins are easily resuspended in 2 D sample solution The procedure can be completed in less than one hour The overall quality of protein separation using 2 D Clean Up Kit has been shown to be superior to that of samples prepared by precipitation with acetone 54 Preparation of protein samples with the kit reduces horizontal streaking improves spot resolution and increases the number of spots detected compared with samples treated by other means Fig 9 and Table 9 pH 4 7 gt fie gt untreated treated with 2 D Clean Up Kit Fig 9 2 D Clean Up Kit eliminates horizontal streaking caused by residual SDS Sample Rat liver extracted with 4 SDS 40 mM Tris base First dimension Approximately 20 ug rat liver protein 7 cm
281. two main types of variation System variation may arise due to differences in electrophoretic conditions between different gels user to user variation or poor performance of the image analysis software This variation can be controlled by the inclusion of an internal standard within each gel and has also been minimized by development of the co detection algorithm within DeCyder 2 D Differential Analysis Software Inherent biological variations are differences that arise between different individuals cell cultures etc These cannot be removed from the analysis but can be accurately measured and therefore differentiated from the system variation Inherent biological variation must be considered if genuine induced biological changes biologically significant changes that arise as a consequence of the test conditions are to be identified It is strongly advised that biological replicates such as multiple cultures be incorporated into the experimental design The more biological replicates included in the experiment the more likely that inherent biological variation is taken into account enabling a reliable measure of significant induced biological change Since the system variation with Ettan DIGE system is low due to the internal standard and analysis method biological variation will far exceed the system variation and gel replicates are therefore not necessary Experimental setup To maximize the benefits of Ettan DIGE system an internal
282. ucing agents are frequently included in the sample preparation solution to break any disulfide bonds present and to maintain all proteins in their fully reduced state The most commonly used reductant is dithiothreitol DTT at concentrations ranging from 20 to 100 mM Dithio erythreitol DTE is similar to DTT and can also be used as a reducing agent Originally 2 mercaptoethanol was used as a reductant but higher concentrations of this reductant are required and inherent impurities may result in artifacts 62 More recently the non thiol reductant tributyl phosphine TBP at a concentration of 2 mM has been used as a reductant for 2 D samples 63 However due to the limited solubility and instability of TBP in solution a thiol reductant such as DTT must also be added to maintain proteins in their reduced state through rehydration and first dimension IEF Reductants should be added directly before use Use of DeStreak Reagent is recommended for basic proteins See section 2 6 2 for details Solubilizing agent Carrier ampholytes or IPG Buffer up to 2 v v can be included in the sample solution They enhance protein solubility by minimizing protein aggregation due to charge charge interactions In some cases buffers or bases e g 40 mM Tris are added to the sample solution This is done when basic conditions are required for full solubilization or to minimize proteolysis However introduction of such ionic compounds can result in first dime
283. ultiple Immobiline DryStrip gels of the same pH range are to be used Carrier ampholytes are added to single aliquots of the stock solution when the same stock solution will be used with different pH range Immobiline DryStrip gels EF Tracking dye bromophenol blue allows IEF progress to be monitored during the protocol If the tracking dye does not migrate toward the anode no current is flowing Note the dye migrates to the end of the strip well before the sample is focused cr Sample can be applied by including it in the rehydration solution Up to 1 mg of sample per strip dependent on the length of the strip and the pH range can be diluted or dissolved in rehydration solution prior to IEF The amount of sample required is dictated in part by the detection or visualization method used For example radiolabeling requires a very small amount of sample whereas Coomassie blue staining requires larger sample amounts 54 80 6429 60 AD 2 6 2 Using DeStreak Rehydration Solution Nonspecific oxidation of protein thiol groups is a common problem during 2 D electrophoresis especially at pH gt 7 In the resulting protein map this problem manifests as horizontal streaks and extra spots DeStreak Reagent and DeStreak Rehydration Solution act to transfer thiol groups in proteins to stable disulfide groups thus preventing nonspecific oxidation This will reduce streaking between spots in the protein map especially in the pH range 7 11 and also simpl
284. ume of wash buffer must be at least 10 fold greater than the distilled deionized water added in step 9 Add 5 ul wash additive use only 5 ul wash additive regardless of the original sample volume Vortex until the pellet is fully dispersed Note The protein pellet will not dissolve in the wash buffer Incubate the tubes at 20 C for at least 30 min Vortex for 20 30 s once every 10 min At this stage the tubes can be stored at 20 C for up to one week with minimal protein degradation or modification 2 Centrifuge the tubes at 8000 x g for 10 min 80 6429 60 AD 13 Carefully remove and discard the supernatant A white pellet should be visible Allow the pellet to air dry briefly no more than 5 min EF Do not over dry the pellet If it becomes too dry it will be difficult to resuspend 14 Resuspend each pellet in rehydration solution for first dimension IEF The volume of rehydration solution used can be as little as 1 20 of the volume of the original sample See next section for examples of rehydration solutions and volumes appropriate for different applications Vortex the tube for 30 s Incubate at room temperature Vortex or aspirate and dispense using a pipette to fully dissolve Er If the pellet is large or too dry it may be difficult to resuspend fully Sonication can speed resuspension 15 Centrifuge the tubes at 8000 x g for 10 min to remove any insoluble material and to reduce any foam The supernatant may b
285. up the gel solution without TEMED or ammonium persulfate Note An optional deaeration step may be performed at this point To do so make up the solution in a vacuum flask Add a small magnetic stirring bar Stopper the flask and apply a vacuum for several minutes while stirring on a magnetic stirrer Just before casting the gel add TEMED and 10 ammonium persulfate Gently swirl the flask to mix being careful not to generate bubbles Immediately pour the gel 80 6429 60 AD 81 5 Pour and prepare the gel Fill the gel cassette to 5 10 mm below the top no stacking gel layer is required Overlay each gel with a layer of water saturated 1 butanol 1 0 ml immediately after pouring to minimize gel exposure to oxygen and to create a flat gel surface After allowing a minimum of 2 h for polymerization remove the overlay and rinse the gel surface with gel storage solution see appendix solution L An alternative to using water saturated 1 butanol to overlay the gels after casting is to spray the edges of the cassettes using a 0 1 w v SDS water solution using a plant sprayer such that the edges are covered by just a few millimeters This technique helps to avoid curved edges on the gels 6 Storage of unused gels Gels not used immediately can be stored for future use at 4 C for up to two weeks Gel storage solution see appendix solution L is pipetted over the top gel surface and the gel cassette is sealed with flexible paraffin f
286. urea reduces electroendosmosis and improves transfer of proteins from the first to the second dimension 3 Dithiothreitol DTT preserves the fully reduced state of denatured unalkylated proteins Sodium dodecyl sulfate SDS denatures proteins and forms negatively charged protein SDS complexes see section 3 2 lodoacetamide alkylates thiol groups on proteins preventing their reoxidation during electrophoresis Protein reoxidation during electrophoresis can result in streaking and other artifacts lodoacetamide also alkylates residual DTT to prevent point streaking and other silver staining artifacts 80 lodoacetamide is introduced in a second equilibration step The second equilibration with iodoacetamide minimizes unwanted reactions of cysteine residues i e when mass spectrometry is to be performed on the separated proteins Tracking dye bromophenol blue allows monitoring of the progress of electrophoresis 80 6429 60 AD 71 3 1 2 Equilibrating Immobiline DryStrip gels w The second dimension vertical gel must be ready for use prior to Immobiline DryStrip gel equilibration If not using the DryStrip gel immediately refer to section 2 8 3 for preservation guidelines Protocol Equilibration is carried out in a two step process using tubes and volume of equilibration solution as specified in Table 22 Preparatory steps 1 Place the IPG strips in individual tubes with the support film toward the tube wall 2 Prepare an appropria
287. uum for several minutes while stirring on a magnetic stirrer Just before casting the gel add TEMED and 10 ammonium persulfate Gently swirl the flask to mix being careful not to generate bubbles Immediately pour the gel Pour and prepare the gel Fill the gel cassette to 5 10 mm below the top no stacking gel layer is required Overlay each gel with a layer of water saturated 1 butanol 0 3 ml immediately after pouring to minimize gel exposure to oxygen and to create a flat gel surface After allowing a minimum of 2 h for polymerization remove the overlay and rinse the gel surface with gel storage solution see appendix I solution L Do not allow the overlay of water saturated 1 butanol to remain on the gel for more than 2 3 h If leaving the gel for a longer period of time replace the 1 butanol with an overlay of running buffer Storage of unused gels Gels not used immediately can be stored at 4 C for up to two weeks Gel storage solution see appendix solution L is pipetted over the top gel surface and the gel cassette is sealed with flexible paraffin film Alternatively the gel cassettes can be stored fully immersed in gel storage solution For further information on the preparation of second dimension vertical SDS slab gels refer to the user manuals for the respective electrophoresis system and multiple gel caster Table 29 Volumes required per vertical gel miniVE SE 260 and SE 600 Ruby systems Casting sy
288. ved detection and identification of low abundance human 18 1151 35 bronchoalveolar lavage fluid BALF proteins using 2 D electrophoresis and Ettan MALDI ToF mass spectrometry application note mproved spot resolution and detection of proteins in 2 D electrophoresis 18 1150 23 using 24 cm Immobiline DryStrip gels application note Multiple mini format 2 D electrophoresis using Hoefer SE 600 Standard 80 6445 94 Vertical Electrophoresis Unit application note Multiple mini format 2 D electrophoresis Multiphor II Flatbed 80 6443 47 application note PlusOne sample preparation kits and reagents brochure 80 6487 74 Protein analysis using the power of 2 D electrophoresis brochure 18 1124 82 Protein electrophoresis technical manual 80 6013 88 Proteomics in Practice A Laboratory Manual of Proteome Analysis 18 1164 45 Westermeier R and Naven T Wiley VCH Verlag GmbH Weinheim 2002 Typhoon Variable Mode Imager data file 63 0048 48 User manuals Ettan DIGE 18 1164 40 DeCyder 2D 7 0 User Manual 28 9414 47 Ettan DALTsix 80 6492 49 Ettan Ettan Gel 12 5 and Ettan DALT Buffer Kit 71 5019 56 Ettan IPGphor 3 11 0034 58 miniVE 80 6420 86 Multiphor II Electrophoresis System 18 1103 43 SE 260 80 6291 95 SE 600 Ruby 80 6353 79 Many of these items can be downloaded from the Literature section on www gehealthcare com 80 6429 60 AD 149 150 80 6429 60 AD Ordering information Product Quantity Code No Samp
289. vides a protocol for use Table 8 lists some of the precipitation techniques that can be used If sample preparation requires precipitation typically only one precipitation technique is employed Table 8 Precipitation procedures Precipitation method General procedure Limitations Ammonium sulfate precipitation Salting out In the presence of high salt concentrations proteins tend to aggregate and precipitate out of solution Many potential contaminants e g nucleic acids will remain in solution Prepare protein so that the final concentration of the protein solution is gt 1 mg ml in a buffer solution that is gt 50 mM and contains EDTA Slowly add ammonium sulfate to the desired percent saturation 44 and stir for 10 30 min Pellet proteins by centrifugation Many proteins remain soluble at high salt concentrations so this method is not recommended when total protein representation is desired This method can however be used for prefractionation or enrichment Residual ammonium sulfate will interfere with IEF and must be removed 45 See section 1 5 on removal of salts TCA precipitation TCA trichloroacetic acid is a very effective protein precipitant TCA is added to the extract to a final concentration of 10 20 and the proteins are allowed to precipitate on ice for 30 min 46 Alternatively tissue may be homogenized directly into 10 20 TCA 35 47 This approach limits proteolys
290. visable only if sample solubility is a problem High conductivity can also arise from the use of poor quality urea or other denaturants Urea is also prone to decomposing to charged breakdown products Higher conductivity salts and ionic impurities in the sample can raise the conductivity of the strip Shorter length IPG strips e g 7 cm strips will not reach 8000 V The distance between the electrodes is shorter so that the voltage gradient V cm required to reach the 50 pA current limit is reached at a lower overall voltage Sample leaks from cup Incorrect cup placement Check that the feet of the cups are resting on the bottom of the manifold channel Check for correct positioning of sample cup arms Check that the feet of the cups are not resting on a centering protrusion in the channel ncorrect strip placement Check that the strip is centered inside of the channel Sparking or burning in the Immobiline DryStrip gels Current limit setting is too high Do not exceed the maximum recommended setting of 50 yA per Immobiline DryStrip gel mmobiline DryStrip gel is not fully rehydrated mmobiline DryStrip gels dried out during IEF Ensure that the Immobiline DryStrip gels are rehydrated with a sufficient volume of rehydration solution Remove any large bubbles trapped under the Immobiline DryStrip gel after placing it on rehydration solution Always apply Immobiline DryStrip Cover Fluid to
291. we m gt KON t Sous gt tou at Hual Fig 22 Ettan IPGphor 3 Control Software For detailed instructions on installation and usage of this software refer to the Ettan IPGphor 3 Control Software user manual 2 3 2 Ettan IPGphor 3 Manifold Ettan IPGphor 3 Manifold is an accessory for first dimension IEF of proteins on IPG strips The Manifold is designed to handle IEF and subsequent equilibration for up to 12 IPG strips All strips in a given run must be of the same length The Manifold can accommodate all IPG strip sizes from 7 to 24 cm It comes with a complete set of accessories for 10 full runs of 12 strips each It is also compatible with the first generation Ettan IPGphor see Ettan IPGphor user manual Cup based sample application can improve protein focusing patterns particularly in basic IPG strips and the Manifold accommodates either anodic or cathodic loading Each cup can hold sample volumes of up to 150 ul Under conditions where substantial water transport electroendosmosis accompanies focusing such as with basic strips or with protein loads in excess of 1 mg the face up mode frequently yields improved resolution Running strips gel side up has a number of advantages over the use of regular Strip Holders 48 80 6429 60 AD e Itis easy to apply filter wicks at the electrodes With preparative loads there is a more pronounced movement of water due to electroendosmotic effects resulting from the extra proteins and pot
292. with a maximum of 600 V or 1 A The power supply unit also controls the temperature of the tank using Peltier elements The operating temperature range of Ettan DALTtwelve system is 10 50 C Gel caster Both Ettan DALTsix and Ettan DALTtwelve systems include gel casters to prepare lab cast gels Separator sheets are sandwiched between the gel casting cassettes for easy removal from the caster following gel polymerization Removable front plates allow for simplified loading and removal of the gel cassettes DALTsix Gel Caster accommodates either six 1 0 mm or six 1 5 mm gel cassettes with separator sheets Fewer gels can be cast by inserting blank cassettes to minimize the volume of casting solution DALTtwelve Gel Caster allows fourteen 1 mm thick gels and thirteen 1 5 mm thick gels to be cast at one time Fewer gels can be cast by inserting blank cassettes to occupy volume not required The caster has a unique hydrostatic chamber to add a displacement solution allowing for volume changes of the solution during polymerization and to produce multiple gels cast to the same height Gradient maker DALTsix Gradient Maker is used in combination with DALTsix Gel Caster for 1 mm thick and 1 5 mm thick gels and with DALTtwelve Gel Caster for 1 mm thick gels It is designed to produce linear gradients of solutions in the volume range 200 1000 ml The gradient maker can be used to form convex or concave exponential gradients by the addition of a one holed rub
293. with minimum user intervention e Spot detection e Background subtraction e In gel normalization e Gel artifact removal e Gel to gel matching e Statistical analysis DeCyder 2 D software utilizes the inclusion of an internal standard within each gel by performing gel to gel matching on the standard samples only The presence of the same standard sample on every gel enables accurate normalization of the individual samples decreasing gel to gel and software analysis variation Differences in expression of less than 10 with over 95 confidence can be achieved within minutes In conjunction with CyDye DIGE Fluor dyes DeCyder 2 D software allows gel analysis using different experimental designs with various degrees of complexity A simple control treated experiment through to a multi factorial experiment addressing factors such as dose and time can be performed in a single analysis The DeCyder 2 D software suite consists of several modules e Batch Processor For automated detection quantitation matching and comparison of multiple gels used with Ettan DIGE system e Differential in gel analysis DIA For co detection background subtraction normalization and quantitation of spots in an image pair e Biological variation analysis BVA For matching multiple gels for comparison and statistical analysis of protein abundance changes e XML Toolbox For exporting spot data from DIA or BVA modules for further analysis For further inf

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