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2-D Electrophoresis

1. 80 6429 60 Edition AB 2 D Electrophoresis using immobilized pH gradients Principles and Methods Amersham g e Biosciences sy TI Antibody Purification Handbook 18 1037 46 The Recombinant Protein Handbook Protein Amplification and Simple Purification 18 1142 75 Protein Purification Handbook 18 1132 29 lon Exchange Chromatography Principles and Methods 18 1114 21 Affinity Chromatography Principles and Methods 18 1022 29 Hydrophobic Interaction Chromatography Principles and Methods 18 1020 90 Gel Filtration Principles and Methods 18 1022 18 Handbooks from Amersham Biosciences Reversed Phase Chromatography Principles and Methods 18 1134 16 Expanded Bed Adsorption Principles and Methods 18 1124 26 Chromatofocusing with Polybuffer and PBE 50 01 022PB Microcarrier cell culture Principles and Methods 18 1140 62 Percoll Methodology and Applications 18 1115 69 Ficoll Paque Plus For in vitro isolation of lymphocytes 18 1152 69 GST Gene Fusion System Handbook 18 1157 58 2 D Electrophoresis using immobilized pH gradients Principles and Methods 80 6429 60 2 D Electrophoresis Principles and Methods Tom Berkelman and Tirra Stenstedt with contributions from Bengt Bjellqvist Nancy Laird Michael McDowell Ingmar Olsson Reiner Westermeler Preface e o et Proteomics is the large scale screening of the proteins of
2. The top surface of the gel has been damaged during application of the IPG strip Bubbles between the gel and the glass plate Liquid between the gel and the glass plate Interfering substances in the first dimension There is an unfilled gap between the gel and one of the spacers Precast gel cassette s not properly closed Bubbles between the gel and the glass plate Liquid between the gel and the glass plate Interfering substances in the first dimension Bubble between IPG strip and top surface of second dimension gel continues on following page Remedy Ensure that all 12 slots in the sealing assembly are occupied Do not pour more than the suggested volume 7 5 into the lower reservoir Ensure that the level of the anode buffer does not come above the sealing assembly when the electrophoresis 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 the 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 Degas the gel solution or increase the amount _ of ammonium persulfate and TEMED by 50 Immediately after pouring the
3. General procedure Prepare protein so 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 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 proteolysis and other protein modifications Centrifuge and wash pellet with acetone or ethanol to remove residual TCA Add at least 3 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 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 Proteins in the sample are extracted into water or buffer saturated phenol Proteins are precipitated from the phenol phase with 0 1 M ammonium acetate in methanol The pellet is washed several times with ammoniu
4. Remedy Be sure that the sample is completely and Stably solubilized Note Repeated precipitation resolubilization cycles produce or increase horizontal streaking See section 1 5 Composition of the sample solution for general guidelines for sample solubilization Increase the concentration of the solubilizing components in the rehydration solution See section 2 4 IPG strip rehydration solution Increase concentration of IPG Buffer Modify sample preparation to limit these contaminants See section 1 4 Removal of contaminants that affect 2 D results Reduce salt concentration to below 10 mm by dilution or desalt the sample by dialysis Precipitation with TCA and acetone and Subsequent resuspension is another effective desalting technique that removes lipids nucleotides and other small molecules Note Specific and non specific losses of proteins can occur with dialysis gel chromatography and precipitation resuspension of samples If the sample preparation cannot be modified the effect of ionic impurities can be reduced by modifying the IEF protocol Limit the voltage to 100 150 V for 2 h then resume a normal voltage step program This pre step allows the ions in the sample to move to the ends of the IPG strip 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 concentrati
5. 1357 1365 1993 Gorg A Boguth G Obermaier C Harder A Weiss W 2 D electrophoresis with immobilized pH gradients using IPGphor isoelectric focusing system Life Science News 1 4 6 1998 Ibel K May R P Kirschner K Szadkowski H Mascher E Lundahl P Protein decorated micelle structure of sodium dodecyl sulfate protein complexes as determined by neutron scattering Eur J Biochem 190 311 318 1990 T2 Tas 74 is 76 Tra 78 TA 80 81 82 83 Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227 680 685 1970 Sch gger H 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 Postel W Weser J G nther S Strahler J R Hanash S M Somerlot L 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 Wilm M Vorm O Mann M Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels Anal Chem 68 850 858 1996 Neuhoff V Stamm R Eibl H Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels A systematic analysis Electrophoresis 6 427 448 1985 Yan J X Wa
6. 1988 Flengsrud R Kobro G A method for two dimensional electrophoreisis of proteins from green plant tissues Anal Biochem 177 33 36 1989 Matsui N M Smith D M Clauser K R Fichmann J Andrews L E Sullivan C M Burlingame A L Epstein L B 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 Kamo M Kawakami T Ohki Y Two dimensional electrophoresis of plant proteins and standardization of gel patterns Electrophoresis 17 855 865 1996 Gorg A Obermaier C Boguth G Csordas A Diaz J J Madjar J J Very alkaline immobilized pH gradients for two dimensional electrophoresis of ribosomal and nuclear proteins Electrophoresis 18 328 337 1997 Usuda H 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 Musante L Candiano G Ghiggeri G M Resolution of fibronectin and other uncharacterized proteins by two dimensional polyacryamide electrophoresis with thiourea J Chromat 705 351 356 1997 91 92 DAN 56 57 58 32 60 61 62 63 64 65 66 67 68 69 70 71 Pasquali C Fialka I Huber L A
7. 3 3 5 Insert the precast gel cassettes into the Ettan DALTtwe ve Separation Unit 1 Insert the gel cassettes Fig 30 When the electrophoresis buffer has reached the desired temperature insert loaded gel cassettes starting at the back of the separation unit ensuring that the IPG strips are in place Push blank cassette inserts into any unoccupied slots When all 12 slots are occupied the buffer level should be slightly below the level of the gaskets O S ci If necessary add distilled or deionized water to bring the level of the lower anode buffer to this level or drain any excess anode buffer that is in the upper chamber The slight dilution of the anode buffer with extra distilled or deionized water will not affect the results Note Cassette insertion is aided by spraying the gel cassettes with a mist of SDS electrophoresis buffer prior to Insertion 2 Pour diluted 1x cathode buffer into the tank to the fill line Note Some of the diluted cathode buffer may drip through the gasket and mix with the anode buffer during the run This mixing effect will not affect performance or results EE Fig 29 Sealing the IPG strip in place using Fig 30 Inserting Precast Gel Cassettes into the Ettan treated agarose sealing solution DALTtwelve Separation Unit 3 3 6 Electrophoresis conditions Table 20 lists the recommended conditions for the Hoefer miniVE SE 260 SE 600 and Ettan DALTtwelve Electrophoresis is performed at consta
8. 34 38 Proteases are less active at lower temperatures so sample preparation at as low a temperature as possible is recommended In addition proteolysis can often be inhibited by preparing the sample in the presence of tris base sodium carbonate or basic carrier ampholyte mixtures These approaches alone are often sufficient protection against proteolysis However some proteases may retain some 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 Table 6 lists common protease inhibitors and the proteases they inhibit For more compre hensive discussions of protease inhibition see references 15 31 and 39 43 Table 6 Protease inhibitors Protease inhibitor Effective against Limitations PMSF Phenylmethylsulfony fluoride PMSF is an irreversible PMSF rapidly becomes inactive in aqueous Most commonly used inhibitor inhibitor that inactivates solutions Prepare just prior to use Use at concentrations up to 1 mM e serine proteases PMSF may be less effective in the presence of e some cysteine proteases thiol reagents such as DTT or 2 mercapto ethanol This limitation can be overcome by disrupting the sample into PMSF containing solution lacking thiol r
9. 72 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 new buffer system based on piperidinopropionamide PPA which combines long shelflife with the high separation pH of the Laemmli system 57 666 66 amp Other buffer systems can also be used particularly the Tris tricine system of Schagger and von Jagow 73 for resolution of polypeptides in the M below 10 000 ExcelGel precast gels for second dimension SDS PAGE on the Multiphor II flatbed system utilize a different Tris tricine buffer system 3 2 IPG strip equilibration The equilibration step saturates the IPG strip 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 Note Equilibration is always performed immediately prior to the second dimension run never prior to storage of the IPG strips at 40 C or lower 3 2 1 Equilibration solution components Equilibration introduces reagents essential for the second dimension separation Equilibration buffer 50 mM Tris HCl pH 8 8 maintains IPG strip pH in a range appropriate for electrophoresis Urea 6 M
10. Al203 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 to disrupt cell suspensions or relatively soft tissues Blenders or other motorized devices can be used for larger samples Homogenization is rapid and poses little danger to proteins except by the proteases that may be liberated upon disruption Glass bead homogenization 23 24 33 The abrasive action of the vortexed beads Cell suspensions Suspend cells in an equal volume of chilled lysis breaks cell walls liberating the cellular microorganisms solution and place into a sturdy tube Add 1 3 g contents of chilled glass beads per gram of wet cells Vortex for 1 min and incubate cells on ice 1 min Repeat vortexing and chilling two to four times 1 2 Protection against proteolysis When cells are lysed proteases are often liberated or activated Degradation 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
11. Choices for second dimension SDS PAGE Multiphor Il Electrophoresis Unit flatbed system 24 5 x 11 cm or 24 5 x 18 cm gels Choice Factors e Precast gels available ExcelGel SDS 12 5 24 5 x 11 cm ExcelGel SDS XL 12 14 24 5 xX 18 em e Relatively rapid 4 h or less for electrophoresis e High resolution e All available IPG strip lengths can be used Fig 3 Multiphor II flatbed system Hoefer miniVE or SE 260 mini vertical 8 x 9 cm gels Choice Factors e Rapid 1 2 h electrophoresis e Best for 7 cm IPG strips Fig 4 Hoefer miniVE Hoefer SE 600 or Ruby standard vertical 14 or 16 x 15 cm gels Choice Factors e 2 5 h electrophoresis fash T e Intermediate separation 16 cm gel length e Intermediate throughput up to four gels simultaneously Best for 13 cm IPG strips Fig 5 Hoefer SE 600 10 Table 1 Equipment choices for 2 D electrophoresis continued Choices for second dimension SDS PAGE Ettan DALT twelve Large Format Vertical System 26 x 20 cm gels Choice Factors e 4h to overnight electrophoresis e Integrated system with very efficient Peltier temperature control e Precast gels with stable buffer system cast on film support available Ettan DALT Gel 12 5 26 x 20 cm 1 mm thickness e Highest resolution 26 x 20 cm gel size e Highest possible protein capacity e High throughput up to 12 gels simultaneously e Best for 18 cm and 24 cm
12. DTT Bromophenol Blue DryStrip Cover Fluid First dimension Immobiline DryStrip Reswelling Tray for 7 18 cm IPG strips Immobiline DryStrip Reswelling Tray for 7 24 cm IPG strips Multiphor Il Immobiline DryStrip Kit focusing system and accessories Multiphor Il Electrophoresis Unit MultiTemp Ill Thermostatic Circulator 115 V MultiTemp Ill Thermostatic Circulator 230 V Immobiline DryStrip Kit Sample cups IEF electrode strips CleanGel electrode strips EPS 3501 XL Power Supply Ettan IPGphor Isoelectric Focusing Unit and accessories Ettan I PGphor Isoelectric Focusing Unit Order Strip Holders separately Ettan PGphor Protocol Guide IEF electrode strips Strip Holders for use with Immobiline DryStrip and Ettan IPGphor Isoelectric Focusing Unit 7 cm 11 cm 13 cm 18 cm 24 cm Ettan IPGphor Cup loading Strip Holder 7 24cm Electrode Cup Loading Strip Holder Quantity Code No 50 samples 80 6483 37 500 assays 80 6483 56 50 samples 80 6484 51 50 samples 80 6484 70 50 samples 80 6483 75 50 samples 80 6483 94 50 samples 80 6484 13 50 samples 80 6484 32 500 g 17 1321 01 500 g 17 1319 01 lg 17 1314 01 500 ml 17 1315 01 lg 17 1318 01 10g 17 1329 01 11 17 1335 01 80 6371 84 80 6465 32 18 1018 06 18 1102 77 18 1102 78 18 1004 30 60 pk 18 1004 35 100 pk 18 1004 40 12 pk 18 1035 33 19 3500 01 80 6414 02 80 6415 73 100 pk 18 1004 40 1 pk 80 6416 87 6 pk 80 6416 11 1 pk 80 6417 06 6 pk 80 641
13. For a general review of protein solubilization for electrophoretic analysis see reference 15 For the first experiments with an unknown sample the following most frequently employed default sample solutions are proposed Dissolve proteins in e 8 M urea 4 CHAPS 60 mM DTT 2 Pharmalyte 3 10 0 002 bromophenol blue To solubilize large and more hydrophobic proteins the following procedure is recommended e 7 M urea 2 M thiourea 4 CHAPS 60 mM DTT 2 Pharmalyte pH 3 10 0 002 bromophenol blue 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 e Grind tissue in mortar and pestle with liquid nitrogen Suspend powder in 10 TCA 0 3 DTT in acetone Keep at 18 C overnight and centrifuge Wash pellet with acetone Dry and resuspend in 9 M urea 2 CHAPS 1 DTT 2 Pharmalyte 3 10 52 63 New kits for mild protein precipitation quick dialysis without protein loss and non interfering protein assays have been introduced by Amersham Biosciences For appropriate sample loads see Table 12 on page 36 Chapter 2 First dimension Isoelectric Focusing IEF 2 0 First dimension isoelectric focusing overview Amersham Biosciences offers two different systems for the first dimension separation the
14. For an overview of precipitation techniques see references 17 18 and 44 1 4 Removal of contaminants that affect 2 D results Non protein impurities in the sample can interfere with separation and subsequent visuali zation of the 2 D result so sample preparation can include steps to rid the sample of these substances Table 8 lists contaminants that affect 2 D results and techniques for their removal Reference 15 provides further discussion on the removal of interfering substances Salt contamination is the most frequent cause of insufficient focusing of protein spots Table 8 Contaminants that affect 2 D results Contaminant Salts residual buffers and other charged small molecules that carry over from sample preparation Endogenous small ionic molecules nucleotides metabolites phospholipids etc continues on following page Reason for removal 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 result causing one end of the strip to dry out and the other to swell Salt in the IPG 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 re
15. S C 3H or in the case of phosphoproteins P For autoradiographic detection the gel is simply dried and exposed to X ray film or for quicker results and superior dynamic range of quantification 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 the most 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 assured specifically for the silver staining application The PlusOne Silver Staining Kit Protein combines superior sensitivity with ease of use By omitting glutardialdehyde from the sensitizer and formaldehyde from the silver nitrate solution the method becomes compatible with mass spectrometry analysis 75 however at the expense of sensitivity When staining Ettan Dalt precast gels with PlusOne Silver Staining Kit Protein a modified staining protocol should be used For details of the modified protocol see Appendix IIT Optimized silver staining of precast DALT 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 tha
16. The buffers Amersham Biosciences Immobiline reagents are a set of well characterized molecules each with a single acidic or basic buffering group linked to an acrylamide monomer The general structure of Immobiline reagents is CH5 H R 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 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 copolymerize with the acrylamide and bisacrylamide monomers to form a polyacrylamide gel Figure 9 is a graphic representation of the polyacrylamide matrix with attached buffering groups Fig 9 Immobilized pH gradient polyacrylamide gel matrix showing attached buffering groups 29 30 For improved performance and simplified handling the IPG gel is cast onto a plastic backing The gel is then washed to remove catalysts and unpolymerized monomers which could otherwise modify proteins and interfere with separation Finally the gel is dried and cut into 3 mm wide strips The resulting IPG strips can be rehydrated with a rehydration solution containing the necessary components for first dimension IEE IEF is performed with the IPG strips placed hori
17. by two dimensional gel electrophoresis using SYPRO dyes Electrophoresis 21 3657 3665 2000 93 94 Additional reading and reference material Code No Application Note Multiple mini format 2 D electrophoresis using precast ExcelGel and Multiphor II flatbed electrophoresis system Application Note Multiple mini format 2 D electrophoresis using Hoefer standard vertical electrophoresis system Data file Immobiline DryStrip visualization of pH gradients Handbook Sample Preparation for Electrophoresis IEF SDS PAGE and 2 D Electrophoresis Principles amp Methods Technical Manual Blot processing with Processor Plus Technical Manual Automated silver and Coomassie staining with Processor Plus Technical Manual Fluorescence Imaging principles and methods Many of these items can be downloaded from www amershambiosciences com Recommended additional consumables Thiourea Fluka Sulfobetains Calbiochem PefaBloc Merck SYPRO Ruby Molecular Probes 80 6443 47 80 6445 94 18 1140 60 80 6429 60 80 6447 27 80 6343 34 63 0035 28 lodoacetamide Fluka Ordering information Product Sample Preparation Sample Grinding Kit 2 D Quant Kit 2 D Clean Up Kit SDS PAGE Clean Up Kit Mini Dialysis Kit 1 kDa cut off up to 250 ul Mini Dialysis Kit 1 kDa cut off up to 2 ml Mini Dialysis Kit 8 kDa cut off up to 250 ul Mini Dialysis Kit 8 kDa cut off up to 2 ml Tris Urea CHAPS Triton X 100 Dithiothreitol
18. j 3 Step and Hold 8 000 3 10 24 6 3 30 Cake Total 4 40 28 6 00 33 6 9 1 Gradient 500 0 01 0 01 0 01 0 01 2 Gradient 4 000 1 30 3 4 2 30 5 6 3 Step and Hold 8 000t 5 20 41 6 5 40 44 3 Total 6 50 45 8 10 50 Narrow 1 Gradient 500 0 01 0 01 0 01 0 01 intervals 2 Gradient 4 000 1 30 3 4 2 30 5 6 3 Step and Hold 8 0007 7 10 56 6 7 40 60 3 Total 8 40 60 10 10 66 6 11 1 Gradient 500 0 01 0 01 0 01 0 01 2 Gradient 4 000 1 30 3 4 2 30 5 6 3 Step and Hold 8 0007 2 40 20 6 2 50 22 3 Total 4 10 24 5 20 28 24 cm 3 10 1 Gradient 500 0 01 0 01 0 01 0 01 3 10 NL 2 Gradient 4 000 1 30 3 4 2 30 5 6 AT 3 Step and Hold 8 000 6 30 51 6 7 40 60 0 3 7 Total 8 00 55 10 10 65 6 9 1 Gradient 500 0 01 0 01 0 01 0 01 2 Gradient 4 000 1 30 3 4 2 30 5 6 3 Step and Hold 8 000 7 10 56 6 11 00 88 0 Total 8 40 60 15 30 106 Narrow 1 Gradient 500 0 01 0 01 0 01 0 01 intervals 2 Gradient 4 000 1 30 3 4 2 30 5 6 3 Step and Hold 80007 12 00 95 6 14 20 114 4 Total 13 30 99 16 50 120 If 1 or 2 IPGbuffer is used decrease the maximum voltage to 3 500 V for 7 cm strips 5 500V for 11 cm strips and 6 S00V for 13 cm strips t This voltage may not be reached within the suggested step duration t Narrow intervals 3 5 4 5 4 0 5 0 4 5 5 5 5 0 6 0 and 5 5 6 7 2 6 6 Troubleshooting JA Table 19 lists possible problems that could be encountered during IEF and how to solve them Table 19 Troubleshooting first dimension IEF Ettan IPGphor Symptom Possible cau
19. pH 7 4 Table 1 Equipment choices for 2 D electrophoresis Choices for first dimension IEF Multiphor II Electrophoresis Unit with Immobiline DryStrip Kit Choice Factors e Multiphor Il Electrophoresis Unit can be used for both first and second dimension separations Fig 1 Multiphor II Electrophoresis Unit e Multiphor Il is a versatile system Its use is not limited to with Immobiline DryStrip Kit IEF with IPG strips from 7 to 24 cm Several different electrophoresis techniques can be performed with the instrument Note EPS 3501 XL Power Supply and Multifemp III Thermostatic Circulator are required to supply power and cool the system Ettan IPGphor Isoelectric Focusing System Choice Factors e Rehydration in Ettan IPGphor Strip Holder Sample application and IEF can be performed overnight unattended e Fewer IPG strip manipulations are required reducing the chance of error e Separations are faster and proteins focus more sharply Fig 2 Ettan IPGphor Isoelectric Focusing C Gl MNEs WOE System e Power supply and temperature control are built into the instrument e Ettan IPGphor Strip Holders in five different lengths from 7 to 24 cm e Ettan IPGphor Cup Loading Strip Holder for all different IPG strip lengths from 7 to 24 cm and for extreme pH gradients e Strip holders are serialized for easy sample tracking Table 1 Equipment choices for 2 D electrophoresis continued
20. proteome analysis Proteome analysis is the analysis of the entire PROTEin complement expressed by a gnOME 6 7 The analysis involves the systematic separation identification and quantification of many proteins simultaneously from a single sample Two dimensional electrophoresis is used in this technique due to its unparalleled ability to separate thousands of proteins simultaneously Two dimensional electrophoresis 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 monitoring therapies drug discovery cancer research purity checks and microscale protein purification This manual describes methods for 2 D electrophoresis using precast IPG strips Immobiline DryStrip gels available from Amersham Biosciences Symbols and abbreviations used in this handbook this symbol indicates general advice which can improve procedures or provide recommendations for action under specific situations this symbol denotes advice that should be regarded as mandatory and gives a warning when special care should be taken this symbol highlights troubleshooting advice to help analyse and resolve difficulties that may occur chemicals buffers and equipment experimental protocol phosphate buffered saline 140 mM NaCl 2 7 mM KCl 10 mM Na HPO 1 8 mM KH PO
21. 154 174 1990 Kawaguchi S I Kuramitsu S Separation of heat stable proteins from Thermus thermophilus HB8 by two dimensional electrophoresis Electrophoresis 16 1060 1066 1995 Teixeira Gomes A P Cloeckaert A Bezard G Dubray G Zygmunt M S Mapping and identification of Brucella melitensis proteins by two dimensional electrophoresis and microsequencing Electrophoresis 18 156 162 1997 Ames G E L Nikaido K Two dimensional gel electrophoresis of membrane proteins Biochemistry 15 616 623 1976 Gorg A Postel W Domscheit A G nther S 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 van den Berg B M Burg H C J Gorg A 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 Theillet C Delpeyroux F Fiszman M Reigner P Esnault R Influence of the excision shock on the protein metabolism of Vicia faba L meristematic root cells Planta 155 478 485 1982 Wolpert T J 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 Bl
22. 200 mm screw cap culture tubes 18 and 24 cm strips in the Equilibration Tubes available from Amersham Biosciences p 53 Table 18 Guidelines for Ettan IPGphor with Cup Loading Strip Holder for broad medium and narrow pH range Immobiline DryStrip gels Voltage gradient and step and hold mode 50 pA IPG strip 0 5 IPG buffer 20 C for IEF Immobiline DryStrip Running conditions for cup loading application method Running conditions for paper bridge application method 54 Length pH range s Step and Voltage Duration Volt hours Duration Volt hours voltage mode V h min kVh h min kVh 7 cm 3 10 1 Gradient 500 0 01 0 01 0 01 0 01 3 10 NL 2 Gradient 4 000 1 30 3 4 2 30 5 6 4 7 3 Step and Hold 5 000 0 45 37 0 30 25 6 11 Total 2 15 7A 3 00 8 0 11 cm 3 10 1 Gradient 500 0 01 0 01 0 01 0 01 4 7 2 Gradient 4 000 1 30 3 4 2 30 5 6 3 Step and Hold 8 0007 1 30 10 6 1 40 12 0 Total 3 00 14 0 4 10 17 6 6 11 1 Gradient 500 0 01 0 01 0 01 0 01 2 Gradient 4 000 1 30 3 4 2 30 5 6 3 Step and Hold 8 000 1 15 8 5 1 30 10 0 Total 2 45 12 4 00 15 6 13 cm 3 10 1 Gradient 500 0 01 0 01 0 01 0 01 3 10 NL 2 Gradient 4 000 1 30 3 4 2 30 5 6 A 3 Step and Hold 8 000 1 50 13 5 2 10 15 2 Total 3 50 17 4 40 20 8 6 11 1 Gradient 500 0 01 0 01 0 01 0 01 2 Gradient 4 000t 1 30 3 4 2 30 5 6 3 Step and Hold 8 000 1 40 11 6 1 50 13 4 Total 3 10 15 4 20 19 0 18 cm 3 10 1 Gradient 500 0 01 0 01 0 01 0 01 3 10 NL 2 Gradient 4 000 1 30 3 4 2 30 5 6
23. 250 mg IAA Tip The subsequent steps of electrophoresis unit preparation insertion of the gel into the precast gel cassette and melting of the Sealing Solution can be performed as the IPG strips are equilibrating 3 3 The Ettan DALTtwe ve system The Ettan DALTtwelve system is designed to handle up to 12 large second dimension gels 26 x 20 cm in a simple efficient and reproducible manner see Fig 6 page 11 Running fewer gels unused slots are filled with the blank cassette inserts Safety interlocks prevent the application of power to the separation unit unless the lid is closed properly and the pump valve is in the circulate position The lid is easily removed for cleaning by sliding it off its hinges Turning the lever at the back of the unit from circulate to drain drains the tank The tempera ture is controlled by Peltier modules attached to the heat exchanger beneath the tank Power Supply Control Unit The Ettan DALTtwelve system is controlled from the Power Supply Control Unit The unit supplies a maximum power output of 200 W with a maximum of 600 V or 1 A The temperature control range is 10 50 C 59 60 3 3 1 Preparation of Ettan DALTtwe ve Separation Unit for electrophoresis 1 Prepare cathode buffer Dilute the cathode buffer included in the Ettan 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 ano
24. Buffer 8 M urea 2 CHAPS 0 002 bromophenol blue Final concentration Amount Urea FW 60 06 8 MI 12g CHAPS 2 wiv 0 5g Bromophenol blue 0 002 w v 50 ul Double distilled H20 to 25 ml DTT and IPG Buffer or Pharmalyte are added just prior to use Add 7 mg DTT per 2 5 ml aliquot of rehydration stock solution See Table 9 page 34 for the appropriate volume of IPG Buffer or Pharmalyte to use For rehydration loading sample is also added to the 2 5 ml aliquot of rehydration solution just prior to use t If necessary the concentration of urea can be increased to 9 or 9 8 M Other detergents Triton X 100 NP 40 and other non ionic or zwitterionic detergents can be used instead of CHAPS Store in 2 5 ml aliquots at 20 C Bromophenol blue stock solution Final concentration Amount Bromophenol blue 1 100 mg Tris base 50 mM 60 mg Double distilled H20 to 10 ml 83 84 C Rehydration stock solution with IPG Buffer 8 M urea 2 CHAPS 0 5 or 2 IPG Buffer 0 002 bromophenol blue 25 ml Final concentration Amount Urea FW 60 06 8 Mi 12 CHAPS 2 w v 0 5g IPG Buffer or Pharmalytet same range as the IPG strip 0 5 v v or 2 v v 125 ul or 500 pl Bromophenol blue 0 002 50 ul 1 solution Double distilled H20 to 25 ml 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 2 5 ml aliquot of rehydration solution
25. IPG strips Fig 6 Ettan DALTtwelve system e Low buffer volume 10 for 12 gels Ettan Spot Picker Robotic system which automatically picks selected protein spots from stained or destained gels using a pick list from the image analysis and transfers them into microplates The gels need to be cast on a plastic backing or glass plate support Fig 7 Ettan Spot Picker 11 The 2 D process begins with sample preparation Proper sample preparation is absolutely essential for a good 2 D result The next step in the 2 D process is IPG strip rehydration IPG strips are provided dry and must be rehydrated with the appropriate additives prior to IEE First dimension IEF is performed on a flatbed system at very high voltages with active temperature control Next strip equilibration in SDS containing buffer prepares the sample for the second dimension separation Following equilibration the strip is placed on the second dimension gel for SDS PAGE The final steps are visualization and analysis of the resultant two dimensional array of spots In summary the experimental sequence for 2 D electrophoresis 1s 1 Sample preparation IPG strip rehydration IEF SDS PAGE 2 3 4 IPG strip equilibration 5 6 Visualization 7 Analysis Equipment choices Different options exist in terms of methods and equipment for IEF and SDS PAGE Table 1 lists the instruments available from Amersham Biosciences For detailed informatio
26. Multiphor II system with associated accessories and the Ettan IPGphor Isoelectric Focusing System For a comparison of these two systems see page 12 A useful first dimension separation requires selecting a first dimension pH range appropriate for the sample as well as a suitable sample application method Choice of immobilized pH gradient is discussed in section 2 2 Sample application methods and their selection are discussed in section 2 3 The first dimension separation procedure involves IPG strip rehydration sample application and isoelectric focusing Preparation of the IPG strip rehydration solution is described in section 2 4 The protocols for IPG strip rehydration sample application and IEF are specific to the first dimension system used and are described in section 2 5 for the Multiphor II system and section 2 6 for the IPGphor Isoelectric Focusing System 2 1 Background to isoelectric focusing IEF IEF is an electrophoretic method that separates proteins according to their isoelectric points pI Proteins are amphoteric molecules they carry either positive negative or zero net charge depending on the pH of their surroundings Fig 8 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 pI is the specific pH at which the net charge of the protein is zero Proteins are positively charged at pH values below their p
27. Orange Protein Gel Stain 500 ul SYPRO Orange Protein Gel Stain 10x50 ul SYPRO Red Protein Gel Stain 500 ul SYPRO Red Protein Gel Stain 10x50 ul SYPRO Tangerine Protein Gel Stain 500 ul SYPRO Gel Stain Starter Kit 1x50 ul of each plus Photographic Filter and Protein Molecular Weight Markers Gel driers Hoefer SE 1200 Easy Breeze Air Gel Drier 115V Hoefer SE 1200 Easy Breeze Air Gel Drier 115V Gel Loading Platform for gels up to 25x21 cm Gel Frame for gels up to 25x21 cm Cellophane Sheets 33x38 cm 50 pk Gel Loading Platform for gels up to 20x20 cm Gel Frame for gels up to 20x20 cm Cellophane Sheets 33x33 cm 50 pk Hoefer GD 2000 Vacuum Gel Drier for gels up to 33x44 cm 115 V Hoefer GD 2000 Vacuum Gel Drier for gels up to 33x44 cm 230 V Cellophane Sheets ImageMaster Image Analysis System ImageScanner Typhoon 8600 Typhoon 9200 Typhoon 9400 Ettan Progenesis Software 1 0 ImageMaster 2D Elite Software ImageMaster 2D Database Software Spot Handling Ettan Spot Picker Ettan Digester Ettan Spot Handling Workstation Mass Spectrometry Ettan MALDI ToF 120 V Ettan MALDI ToF 230 V Code No 80 6445 18 80 6444 23 80 6343 34 80 6468 17 17 0518 01 RPN5801 RPN5802 RPN5803 RPN5804 RPN5805 RPN5811 80 6121 61 80 6121 80 80 6429 41 80 6429 22 80 6430 17 80 6429 22 80 6429 22 80 6121 99 80 6428 84 80 6429 03 80 6117 81 18 1134 45 63 0027 96 Inquire Inquire 18 1154 43 80 6350 5
28. 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 Perdew G H Schaup H W Selivonchick D P The use of a zwitterionic detergent in two dimensional gel electrophoresis of trout liver microsomes Anal Biochem 135 453 455 1983 Rabilloud T Blisnick T Heller M Luche S Aebersold R Lunardi J Braun Breton C 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 Santoni V Molloy M Rabilloud T Membrane proteins and proteomics Un amour impossible Electrophoresis 21 1054 1070 2000 Wilson D L Hall M E Stone G C Rubin R W Some improvements in two dimensional gel electrophoresis of proteins Anal Biochem 83 33 44 1977 Marshall T Williams K M Artifacts associated with 2 mercaptoethanol upon high resolution two dimensional electrophoresis Anal Biochem 139 502 505 1984 Herbert B R Molloy M P Gooley A A Walsh B J Bryson W G Williams K L Improved protein solubility in two dimensional electrophoresis using tributyl phosphine as reducing agent Electrophoresis 19 845 851 1998 Gorg A Obermaier C Boguth G Ha
29. Quantity Pharmalyte 25 ml pH 3 10 pH 5 8 pH 8 10 5 Second dimension 2 D Electrophoresis brochure Hoefer mini vertical units and accessories Hoefer miniVE complete includes basic unit two 10 well 1 0 mm combs and two pairs of 1 0 mm spacers for up to 2 gels glass plate size 10x10 5 cm Spacer 1 0 mm 2 pk Spacer 1 5 mm 2 pk SE 260 Mighty Small II Vertical Unit complete for 2 slab gels SE 235 Mighty Small 4 Gel Caster complete SE 245 Mighty Small Dual Gel Caster Thin fluorescent rulers 2 pk Hoefer Wonder Wedge plate separation tool Hoefer SE 600 vertical units and accessories SE 600 Dual Cooled Vertical Slab Unit for up to 4 gels glass plate size 18x16 cm Spacer 1 0 mm 1 cm wide 2 pk Spacer 1 0 mm 2 cm wide 2 pk Spacer 1 5 mm 1 cm wide 2 pk Spacer 1 5 mm 2 cm wide 2 pk Divider glass plate 18x16 cm notched SE 615 Multiple Gel Caster for 2 to 10 gels glass plate size 18x16 cm Glass plates 18x8 cm 2 pk Divider glass plate 18x8 cm notched Clamp assembly 8 cm 2 pk Spacer 1 0 mm 1 cm wide 8 cm long 2 pk Spacer 1 5 mm 1 cm wide 8 cm long 2 pk Ettan DALT twelve Large Vertical System and accessories Ettan DALT twelve Separation Unit and Power Supply Control Unit 115 VAC Ettan DALT twelve Separation Unit and Power Supply Control Unit 230 VAC Includes Ettan DALT Cassette Removal Tool 2 pk Ettan DALT Buffer Seal Removal Tool 2 pk Order accessories separately Ettan D
30. V 400 mA 200 W EPS 2A200 Power Supply 200 V 2000 mA 200 W EPS 301 Power Supply 300 V 400 mA 80 W EPS 601 Power Supply 600 V 400 mA 100 W EPS 1001 Power Supply 1000 V 400 mA 100 W Thermostatic circulator MultiTtemp Ill Thermostatic Circulator 115 V MultiTemp Ill Thermostatic Circulator 230 V ExcelGel SDS gels ExcelGel SDS 2 D homogeneous ExcelGel SDS XL 12 14 ExcelGel SDS Buffer Strips anode and cathode Quantity 16 pk 6 pk 2 pk 2 p 1 set 2 pieces 6 pk 30 ml total volume 50 ml total volume 100 ml total volume 500 ml total volume 200 pk 6 pk 3 pk 6 each pk Code No 80 6127 88 80 6467 22 80 6467 41 80 6467 60 80 6467 98 80 6474 63 80 6474 82 80 6475 39 80 6475 58 80 6067 65 80 6067 84 17 6002 36 17 6002 50 80 6197 80 80 6197 99 80 6196 09 80 6198 18 18 1018 06 80 6442 90 18 1013 75 80 1129 46 18 1130 04 18 1130 05 80 6406 99 18 1130 01 18 1130 02 18 1130 03 18 1102 77 18 1102 78 80 6002 21 17 1236 01 17 1342 01 Product PlusOne gel casting chemicals and buffers Acrylamide PAGE acrylic acid lt 0 05 Acrylamide PAGE acrylic acid lt 0 05 Acrylamide IEF acrylic acid lt 0 002 Acrylamide IEF acrylic acid lt 0 002 Acrylamide IEF 40 solution Acrylamide PAGE 40 solution N N methylenebisacrylamide N N methylenebisacrylamide 2 solution Agaraose M Agarose NA Glycine Ammonium persulfate TEMED P
31. a rehydration solution containing a zwitterionic or non ionic detergent CHAPS Triton X 100 or NP 40 so the final concentration 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 Treat samples rich in nucleic acids with a protease free DNase RNase mixture to reduce the nucleic acids to mono and oligonucleotides This is often done by adding 0 1 x volume of a solution containing 1 mg ml DNase I 0 25 mg ml RNase A and 50 mM MgCl followed by incubation on ice 33 50 Note The proteins DNase and RNase may appear on the 2 D map Ultracentrifugation can be used to remove large 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 Precipitate the sample in TCA ammonium sulfate or phenol ammonium acetate then centrifuge Ultracentrifugation will remove high molecular weight polysaccharides Employing the same methods used for preventing pr
32. detergents are used Sample solution composition is discussed in section 1 5 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 loss The cells or tissue should be disrupted in such a way as to minimize proteolysis and other modes of protein degradation Cell disruption should be done 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 80 C Do not expose samples to repeated thawing Remove all particulate material by ultracentrifugation Solid particles and lipids must be removed because they will block the gel pores To avoid modification of proteins never heat a sample after adding urea When the sample contains urea it must not be heated over 37 C Elevated temperatures cause urea to hydrolyze to isocyanate which modifies proteins by carbamylation For more specific guidance on preparing samples for application to IPG strips see references 14 16 1 1 Methods of cell disruption Listed in Table 4 and Table 5 are a few standard disruption methods both mechanical and chemical Cell d
33. each IPG strip with 3 ml of DryStrip Cover Fluid to minimize evaporation and prevent urea crystallization 5 Allow the IPG strip to rehydrate Slide the lid onto the Reswelling Tray and allow the IPG strips to rehydrate at room temperature A minimum of 10 h is required for rehydration overnight is recommended If the IPG strips swell unevenly refer to Table 12 on page 36 Note Rehydrate the IPG strips using the Immobiline DryStrip Reswelling Tray If the Immobiline DryStrip Reswelling Tray is not available strips can be rehydrated in the Ettan IPGphor Strip Holder Note Rehydration in the Ettan PGphor Cup Loading Strip Holder is not recommended the channel is too wide for the rehydration volume For rehydration in Ettan IPGphor Strip Holder see page 44 Prepare the Ettan IPGphor Cup Loading Strip Holder 1 Position the strip holder on the Ettan IPGphor platform Due to the high voltage applied to the Ettan IPGphor Cup Loading Strip Holder it is important that it be clean and dry The pointed end of the strip holder should contact the anodic electrode area and the blunt end should contact the cathodic electrode area of the Ettan IPGphor separation platform 2 Transfer the strips to the Ettan IPGphor Cup Loading Strip Holder The strips should be placed face up in the tray with the anodic pointed end of the IPG strip toward the pointed end of the strip holder The strip must be positioned so that the gel overlaps the
34. ends of both plated regions of the strip holder The cathodic end of the IPG strip must be approximately 1 5 cm from the end of the channel and in electrical contact with the cathodic rails via the electrode clips Fig 20 Center the strip down the length of the strip holder channel Protrusions along the sides guide the strip approximately straight although some manual adjustment of the strip may be necessary 3 Overlay Immobiline DryStrip Cover fluid across the surface of the IPG strip It is important to distribute the oil evenly across the IPG strip and down the entire length of the Ettan PGphor Cup Loading Strip Holder Use only enough oil to cover the strip without overfilling 3 5 ml 49 Cup Loading Rail Strip holder YS x Y T N AN area Fig 20 Positioning the IPG strips so that the gels are in electrical contact with the rails via the electrode clips 4 Optional Cut electrode pads from IEF electrode strips about 5 mm long Two pads per strip holder are required Wet the pads with deionized water and blot them almost completely dry Longer pads can be used if desired If using longer pads one end of the pad should overlap the end of the gel on the IPG strip The electrode must contact the other end of the pad Place pads on both ends of the IPG strip Fig 21 Slide an electrode down on top of each pad Depending on the thickness of the IEF pad the electrode may not feel solid on top of the filter paper How
35. gel overlay the surface with water saturated butanol Take care during application of the IPG strip that the gel is not damaged Use the roller to remove any bubbles or excess _ liquid between the gel and the glass plate Ensure that no visible bubbles remain and that the gel adheres firmly to the glass and resists movement Contaminants in the sample can cause distortions or swollen regions in the IPG strip following IEF These distortions can result in turn in disturbances in the second dimension Modify sample preparation to limit these contaminants When sealing the IPG strip into place on top of the gel ensure that some of the sealing solution flows down any gap that may exist _ between the gel and spacer Ensure cassette s are properly closed and repeat the run Use the roller to remove any bubbles or excess liquid between the gel and the glass plate Ensure that no visible bubbles remain and that the gel adheres firmly to the glass and resists movement Contaminants in the sample can cause distortions or swollen regions in the IPG strip following IEF These distortions can result in turn in disturbances in the second dimension Modify sample preparation to limit these contaminants Ensure that no bubbles are trapped between the IPG strip and the top surface of second dimension gel 67 68 Table 25 Troubleshooting vertical second dimension SDS PAGE continued Symptom Possible ca
36. grooves of the aligner in the Immobiline DryStrip tray Place the strips with the pointed acidic end at the top of the tray near the red electrode anode The blunt end Should be at the bottom of the tray near the black electrode cathode Align the IPG strips so the anodic gel edges are lined up 3 Attach the electrode strips Place the moistened electrode strips across the cathodic and anodic ends of the aligned IPG strips The electrode Strips must at least partially contact the gel surface of each IPG strip 4 Position the electrodes Fig 13 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 IPG strips are still aligned in their grooves Fig 12 Positioning IPG strip gels in the Immobiline Fig 13 Alignment of electrodes over IPG strips DryStrip aligner 2 5 3 Sample application by cup loading 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 IPG strips are given in Table 11 on page 36 These values should only be regarded as a rough gu
37. just prior to use t An IPG Buffer Pharmalyte concentration of 0 5 is recommended with the IPGphor and an IPG Buffer Pharmalyte concentration of 2 is recommended with the Multiphor I and Immobiline DryStrip Kit system If necessary the concentration of urea can be increased to 9 or 9 8 M 1 Other detergents Triton X 100 NP 40 and other non ionic or zwitterionic detergents can be used instead of CHAPS Use 125 ul IPG Buffer for a 0 5 concentration and 500 pl IPG Buffer for a 2 concentration tt Use Pharmalyte 3 10 for Immobiline DryStrip 3 10 or 3 10 NL Pharmalyte 5 8 for Immobiline DryStrip 4 7 Store in 2 5 ml aliquots at 20 C D SDS equilibration buffer 50 mM Tris HCl pH 8 8 6 M urea 30 glycerol 2 SDS bromophenol blue 200 ml Final concentration Amount Tris HCl pH 8 8 see solution F 50 mM 10 0 ml Urea FW 60 06 6M 72 07 g Glycerol 87 v v 30 v v 69 ml SDS FW 288 38 2 wiv 4 0g Bromophenol blue 0 002 w v 400 ul of 1 solution Double distilled H20 to 200 ml This is a stock solution Prior to use DTT or iodoacetamide are added See section 3 2 2 Store at 20 C E 30 T 2 6 C monomer stock solution 30 acrylamide 0 8 N N methylenebisacrylamide 200 ml Final concentration Amount Acrylamide FW 71 08 30 60 0 g N N methylenebisacrylamide FW 154 17 0 8 1 6g Double distilled HO to 200 ml Filter solution through a 0 45 pm filter Store at 4 C in the dark F 4x re
38. pH 4 7 and 6 9 to obtain a more detailed overview of the protein distribution For studying the protein pattern in more detail with the highest resolution and sample load narrow pH gradients in 18 and 24 cm strips offered for extremely high resolution are available pH 3 5 4 5 4 5 4 5 5 5 5 6 and 5 5 6 7 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 immobilized pH gradients are given in 67 2 3 Sample application method selection Sample can be applied either by including it in the rehydration solution rehydration loading or by applying it directly to the rehydrated IPG strip via sample cups sample wells or paper bridge Usually rehydration loading is preferable see section 2 4 Advantages to this mode of application include the following e Rehydration loading allows larger quantities of protein to be loaded and separated 65 66 e Rehydration loading allows more dilute samples to be loaded e Because there is no discrete application point this method eliminates the formation of precipitates at the application point that often occur when loading with sample cups The rehydration loading method is technically simpler avoiding problems of leakage that can occur when using sample cups e There are however cases when one might prefer to load the sample fol
39. the lid and carefully remove the electrodes 2 600 50 30 1 10 ExcelGel XL SDS gradient 12 14 1 200 20 20 0 40 Open the lid and carefully remove the electrodes 2 800 40 40 2 40 Remove the IPG strip and the application pieces Then move the cathodic buffer strip forward to cover the area of the removed IPG strip Adjust the position of the cathodic electrode t Electrophoresis is stopped 5 min after the bromophenol blue front has just reached the anodic buffer strip Remove and discard the buffer strips 3 4 4 Troubleshooting Table 27 lists possible problems that could be encountered during second dimension SDS PAGE using the Multiphor II flatbed system and how to solve them Table 27 Troubleshooting second dimension SDS PAGE Multiphor II flatbed system Symptom No current at start of run Dye front curves up smiles at one edge Dye front curves up smiles at both edges Dye front is irregular Buffer strip slides out from under the electrode Possible cause The electrode cable is not plugged in Cathodic buffer strip does not contact the gel at the one edge Inadequate cooling Some dye front irregularity results from the use of IPG Buffer and does not affect results Buffer strips or ExcelGel are old Bubbles under the buffer strip Bubbles under the IPG strip Incorrect electrode placement Remedy Ensure that all cables are properly connected Ensure that the cathodic buffer strip is c
40. the setup Check that strips are in their correct straight position in the DryStrip aligner 5 Apply DryStrip Cover Fluid Once the sample cups are properly positioned pour 70 to 80 ml DryStrip Cover Fluid into the tray to completely cover the IPG strips If the 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 additional DryStrip Cover Fluid to completely cover the sample cups The IPG strips are submerged under a layer of DryStrip Cover Fluid to prevent drying of the IPG strip precipitation of the components of the rehydration solution and diffusion of gas into the IPG strip 6 Apply the sample Fig 15 Apply sample up to 100 ul per IPG strip into the sample cups by pipetting under the surface of the DryStrip Cover Fluid The sample should sink to the bottom of the cup Check for leakage 7 Start IEF Ensure 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 Begin IEF Note 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 non ionic detergents and IPG buff
41. together with glycerol reduces the effects of electroendosmosis by increasing the viscosity of the buffer 4 Electroendosmosis is due to the presence of fixed charges on the IPG strip in the electric field and can interfere with protein transfer from the IPG strip to the second dimension gel Glycerol 30 together with urea reduces electroendosmosis and improves transfer of protein from the first to the second dimension 4 DTT preserves the fully reduced state of denatured unalkylated proteins Sodium dodecyl sulfate SDS denatures proteins and forms negatively charged protein SDS complexes The amount of SDS bound to a protein and therefore the additional negative charge is directly proportional to the mass of the protein Thus electrophoresis of proteins through a sieving gel in the presence of SDS separates proteins on the basis of molecular mass Iodoacetamide 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 74 Ilodoacetamide is introduced in a second equilibration step This step is optional when SDS PAGE is performed in a vertical second dimension system but required when SDS PAGE is performed on a flatbed second dimension system especially when the flatbed separation is to be visualized by silver sta
42. typically complete in 1 to 2 h The use of mini gels for the second dimension of 2 D is ideal when quick profiling is required or when there are relatively few different proteins in the sample For increased throughput and resolution the standard sized SE 600 vertical gel system Fig 5 page 10 is recommended The SE 600 accommodates up to four 16 cm long gels and the built in heat exchanger offers cooling capability 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 IPG strip 1 cm wide spacers are available for the preparation of 16 cm wide gels For maximal resolution reproducibility and capacity the large gel format of the Ettan DALTtwelve system Fig 6 page 11 is recommended Precast large format Ettan DALT gels on film support offer the convenience of ready to use gels The system can accommodate the entire length of an 18 and 24 cm IPG strip plus molecular weight markers and up to twelve gels can be run simultaneously Integrated Peltier temperature control 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 Laboratory technique Always wear gloves when handling IPG strips SDS polyacrylamide gels ExcelGel Buffer Strips and any equipment that these items will contact The use of glov
43. 0 Second dimension SDS PAGE overvieW ccccceceeeeeeceeeeeeeceeeeeeeeeeeeeaeaeseegeeeeeenass 57 5 1 Ba keround to SDS PAGE lt incsscnsastavencassnasntneadesnsansaanesivescianatniacasaeotanateneebesradasatess 57 See PG Sir lo eo IMEI eaaa aa E abalseanatuen tat 58 3 2 1 Equilibration solution components sc cicnancacuieciessvevshcaidiniainasludiavahadavewenundnd aidvads weuaiaseduisinussneulanatixed 58 3 2 2 Equilibration StepS aicawucucevianaymudnon ane n ines iesenenenweusamasaaneasamnamnnnnnsay abana snontiammumullenansntdnasexaeumamaumpns 59 3 3 The Ettan DALT twelve Syster ciseintsiasctcciutinduratycaiiecubevesis moiestbddaehuadgusaesneresSauateaads 59 3 3 1 Preparation of Ettan DALTtwelve Separation Unit for electrophoresis cccccceecseeeeeeeeeeeeeeeeeees 60 3 3 2 Ettan DALT precast gels gene eee nr EEEN OE NEEE EA ee eer 60 3 3 3 Equilibrate the IPG strip ce tatsetccicanuvetsieamtedndadiesmvcnince cantesutaiiacatesiiemeaoadateetmbusesaiiaecidndetneteniseaeipuntaidn taamaiarinied 61 3 3 4 Applying the equilibrated IPG Strip sencanastuncseswauxwanwvaniodseandasneiepasaoarcensinandstrannnbeonetedsmuaxentennes 61 3 3 5 Insert the precast gel cassettes into the Ettan DALTtwelve Separation Unit ccceceeeeeeeeeeeeeees 63 3 3 6 Electrophoresis conditions sncrscetaia etiencectneus nah anuxbenamsaienranicauieesarabaudocinediceehe vtancatundtiansinauduteinee 63 3 3 7 Preparing SDS slab gels vertical SYSTEMS ccccccccecseceeee
44. 6 80 6351 13 18 1145 28 Inquire Inquire 18 1145 00 18 1142 33 CleanGel Ettan Eazy Breeze ExcelGel Hoefer ImageMaster ImageScanner Immobiline IPGphor Multiphor MultiTemp Pharmalyte PlusOne and Typhoon are trademarks of the Amersham Biosciences group Amersham and Amersham Biosciences are trademarks of Amersham plc Coomassie is a trademark of ICI ple SYPRO is a trademark of Molecular Probes Inc Tris and Triton X 100 are trademarks of Rohm amp Haas Tween is a trademark of ICI Americas Inc All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them A copy of these terms and conditions is available on request Amersham Biosciences AB 2001 All rights reserved Amersham Biosciences AB AB Bj rkgatan 30 SE 751 84 Uppsala Sweden Amersham Biosciences Amersham Place Little Chalfont Buckinghamshire HP7 9NA England Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences Sanken Building 3 25 1 Shinjuku ku Tokyo 169 0073 Japan www amershambiosciences com Amersham r e Biosciences Production RAK Design AB
45. 6 30 1 pk 80 6417 25 6 pk 80 6416 49 1 pk 80 6417 44 6 pk 80 6416 68 1 pk 80 6470 07 6 pk 80 6469 88 3 pk 80 6459 43 6 pk 80 6464 94 95 96 Product Quantity Strip Holders for use with Immobiline DryStrip and Ettan IPGphor Isoelectric Focusing Unit continued Sample Cup Ettan IPGphor Cup Loading Strip Holder 50 pk Cleaning solution Ettan IPGphor Strip Holder 950 ml Immobiline DryStrip gels 7 cm pH 3 10 L 12 pk pH 3 10 NL pH 4 7 pH 6 11 11 cm pH 3 10 L 12 pk pH 4 7 pH 6 11 13 cm pH 3 10 L 12 pk pH 3 10 NL pH 4 7 pH 6 11 18 cm pH 3 10 L 12 pk pH 3 10 NL pH 4 7 pH 6 11 pH 6 9 pH 3 5 4 5 pH 4 0 5 0 pH 4 5 5 5 pH 5 0 6 0 pH 5 5 6 7 24 cm pH 3 10 L 12 pk pH 3 10 NL pH 4 7 pH 6 9 pH 3 7 NL pH 3 5 4 5 pH 4 0 5 0 pH 4 5 5 5 pH 5 0 6 0 pH 5 5 6 7 Equilibration Tube Set for up to 12 pk 24 cm IPG strips IPG Buffer 1 ml pH 3 5 5 0 pH 4 5 5 5 pH 5 0 6 0 pH 5 5 6 7 pH 4 7 pH 6 11 pH 3 10 pH 3 10 NL Code No 80 6459 81 80 6452 78 17 6001 11 17 6001 12 17 6001 10 17 6001 34 18 1016 61 18 1016 60 17 6001 35 17 6001 14 17 6001 15 17 6001 13 17 6001 96 17 1234 01 17 1235 01 17 1233 01 17 6001 97 17 6001 88 17 6001 83 17 6001 84 17 6001 85 17 6001 86 17 6001 87 17 6002 44 17 6002 45 17 6002 46 17 6002 47 17 6002 43 17 6002 38 17 6002 39 17 6002 40 17 6002 41 17 6002 42 80 6467 79 17 6002 02 17 6002 04 17 6002 05 17 6002 06 17 6000 86 17 6001 78 17 6000 87 17 6000 88 Product
46. 8 1 12 1 3 3 500 2 05 7 1 Total 3 50 5 00 11 15 Total 3 35 10 3 10 1 300 0 01 0 001 2 3500 1 30 2 9 3 3 500 2 05 2 35 7 1 9 1 Total 3 35 4 05 10 12 13cm 4 7 1 300 0 01 0 001 13cm 6 11 1 300 0 01 0 01 2 3500 1 30 2 9 2 3 500 1 30 2 9 3 3 500 3 45 4 20 13 1 18 1 3 3 500 3 10 11 1 Total 5 15 5 50 16 21 Total 4 40 14 3 10 1 300 0 01 0 001 3 10NL 2 3500 1 30 2 9 3 3 500 3 10 4 00 11 1 14 1 Total 4 40 5 30 14 17 18cm 4 7 1 500 0 01 0 001 18cm 6 11 1 500 0 01 0 01 2 3500 1 30 3 0 2 3 500 1 30 3 0 3 3 500 5 40 7 40 20 27 3 3 500 5 40 20 0 Total 7 10 9 10 23 30 Total 7 10 23 3 10 1 500 0 01 0 001 6 9 1 500 0 01 0 01 3 10NL 2 3500 1 30 3 0 2 3 500 1 30 3 0 3 3 500 4 50 6 20 17 22 3 3 500 12 00 42 Total 6 20 7 50 20 25 Total 13 30 45 Narrow 1 500 0 01 0 001 intervals 2 35007 1 30 3 0 3 3 500 13 20 16 20 47 57 Total 14 50 17 50 50 60 24cm 3 10 1 500 0 01 0 001 24cm 6 9 1 500 0 01 0 01 2 3500 1 30 3 0 2 3 500 1 30 3 0 3 3 500 7 40 10 40 27 37 3 3 500 16 20 57 Total 9 10 12 10 30 40 Total 17 50 60 4 7 1 500 0 01 0 001 3 7 NL 2 3500 1 30 3 0 3 10NL 3 3 500 12 00 16 20 42 47 Total 13 30 17 50 45 60 Narrow 1 500 0 01 0 001 Intervals 2 35007 1 30 3 0 3 3 500 22 00 27 40 77 97 Total 23 30 29 10 80 100 For all Immobiline DryStrip gels Temperature 20 C Current 0 5 mA strip Power 5 W total Kilovolt hour k Vh values are recommended t Program EPS 3501 XL power supply with current check option turned off IPG strip i
47. ALT Precast Gel Cassette Ettan DALT Gel Casting Cassette 1 0 mm Ettan DALT Blank Cassette Insert Roller needed for precast Code No 17 0456 01 17 0453 01 17 0455 01 18 1124 82 80 6418 77 80 6150 11 80 6150 30 80 6149 35 80 6146 12 80 6146 50 80 6223 83 80 6127 88 80 6171 58 80 6179 94 80 6180 70 80 6180 13 80 6180 89 80 6179 18 80 6182 79 80 6186 59 80 6186 78 80 6187 35 80 6443 09 80 6443 28 80 6466 46 80 6466 27 80 6474 82 80 6474 63 80 6466 65 80 6466 84 80 6467 03 80 1106 79 97 98 Product Order accessories separately continued Wonder wedge needed for lab cast Ettan DALTtwe ve Gel Caster Complete Includes 80 6467 41 and 80 6467 60 Ettan DALT Separator Sheets 0 5 mm Ettan DALT Filler Sheets 1 0 mm Ettan DALT Cassette Rack Ettan DALT Buffer Seal Remover Tool Ettan DALT Cassette Removal Tool Ettan DALT Glass Set Ettan DALT LF Glass Set Hoefer DALT Gradient Maker with peristaltic pump 115 V Same as above 230 V Ettan DALT gels and buffer kit Ettan DALT Gel 12 5 homogeneous Ettan DALT Buffer Kit Gradient makers SG 30 Gradient Maker SG 50 Gradient Maker SG 100 Gradient Maker SG 500 Gradient Maker Multiphor Il Multiphor Il Electrophoresis Unit Multiphor Il Buffer Strip Positioner Film remover for electrophoretic transfer IEF sample application pieces Power supplies EPS 3501 Power Supply 3500 V 150 mA 100 W EPS 3501 XL Power Supply 3500
48. CUSINS OVEIVIEW ccccccecceceseeeeeeeeeeeeeauseeseseeeesseeaeens 27 2 4 Background to isoelectric focusing IEF csccesernasesssiarue dene ccsanntecdedunannasedecans tedsaienmaas 27 2 2 Immobilized pH gradient selection cccccececcececeseeeeceeeeeeeecesaseeaeseseeeeaeseseeaensenas Sl 2 3 Sample application Method selection cccccceccececseeececeeeesececeseeeeceseeueeeseseeeeaeees 31 2A IPG Strip renydration SOLUTION cst tinsiiataireudiesiediethavaetadiasancedunaiins MaiiebadiaNiadacnsaint hivinietnieas 33 2 4 1 Components of The rehydration solution idvinsnimsacnesnnecnuneatodedwicanrcarasdduteaniabonerndesmmavaraxtwedensmenteds 33 2 4 2 Rehydration solution preparation cccccscsccececceccecececceceeeeceseesecseseseeeeseeesueeeauseesesseaeseeaeeees 35 2 9 Multiphor Il and Immobiline DryStrip Kit s s sisssassrernarussnnnnnnsdnnnan 55 2 5 1 IPG strip rehydration Immobiline DryStrip Reswelling Tray cccccccccceceeeeeecaeeeeeeeeeeeeeseeeseeeaes 35 2 5 2 Preparing for EF misccagasiaentathstisutsamnectatadeesiabornabheianboiineniardcbeubichinestaidtidaaniataniahiaenmadatniens 37 2 5 3 Sample application by cup loading sisdscacicnsiceconensswnavsernchcs cinusmoavansnamcsienedabesnavsamewnnssanceenniaerinwens 38 2 5 4 Paper bridge loading sisncainicsisasnsnsixsasinitn cedniustinnandanciu duwsiunamontnedtcuide teas dueuiniewamiesniaatandesascmunamespondusaness 39 2 5 5 Isoelectric focusing guidelines scusaictcensucecvn
49. I and negatively charged at pH values above their pI 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 8 The presence of a pH gradient is critical to the IEF technique In a pH gradient under the influence of an electric field a protein will move to the position in the gradient where its net charge is zero A protein with a positive net charge will migrate toward the cathode becoming progressively less positively charged as it moves through the pH gradient until it reaches its pI A protein with a negative net 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 pIs 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 1 000 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 below 1 mA IEF of a given sample in a given electrophoresis system is generally performed for a constant number of Volt hours Volt hour Vh being t
50. Immobiline DryStrip Reswelling Tray 35 36 4 Overlay the IPG strip with DryStrip Cover Fluid Overlay each IPG strip with 3 ml of DryStrip Cover Fluid to minimize evaporation and urea crystallization 5 Allow the IPG strip to rehydrate Slide the lid onto the Reswelling Tray and allow the IPG strips to rehydrate at room temperature A minimum of 10 h is required for rehydration overnight is recommended If the IPG strips swell unevenly refer to Table 12 6 Prepare the Immobiline DryStrip Kit Before removing the IPG strips from the Reswelling Tray prepare the Immobiline DryStrip Kit and the electrode strips as described in section 2 5 2 A and 2 5 2 B Table 11 Suitable sample loads for silver and Coomassie staining using cup loading and rehydration loading Immobiline DryStrip pH 7cm 4 7 6 1 3 1 11 cm 4 7 6 1 3 1 13 cm 4 7 6 1 3 1 18 cm 4 7 6 1 3 1 24 cm 4 7 6 9 3 1 1 O 3 10 NL 2 4 Suitable sample load pg of protein Silver stain 4 8 8 16 10 20 20 40 1 OL 4 8 15 30 30 60 1 O 3 10 NL 8 15 30 60 6 9 narrow interval 60 120 3 10 NL 15 30 7 3 7 45 90 rrow interval 80 170 Na O 3 10 NL 20 40 Coomassie stain 20 120 40 240 10 60 50 300 100 600 20 120 75 450 150 900 40 240 150 900 300 1 500 75 450 200 1 300 400 2 000 100 600 When using cup loading an increased sample concentration will lead to an increased risk of protein p
51. Make up to 250 ml with distilled water Washing Distilled water 4x1 min Developing Sodium carbonate 6 25 g 1 packet 5 mint Formaldehyde 37 100 ul Make up to 250 ml with distilled water Stir vigorously to dissolve sodium carbonate Stop EDTA Nas x H20 3 65 g 1 packet 45 min Make up to 250 ml with distilled water Washing Distilled water 2 x 30 min Preservation Glycerol 87 25 ml 20 min Make up to 250 ml with distilled water The first fixation may be prolonged up to 3 days if desired for convenience t By omitting glutardialdehyde from the sensitizer and formaldehyde from the silver nitrate solution as well as omitting the preservative step the method becomes compatible with mass spectroscopy 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 between 100 pl up to 250 pl 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 1 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 87 88 References is 10 11 12 l3 14 15 16 O Farrell P H High resolution two dimensional electrop
52. Samples should be applied using cup loading at the acidic end of the IPG strip 2 5 7 Running a protocol Ensure that the electrodes in the Immobiline Dry Strip 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 IEE As isoelectric focusing proceeds the bromophenol blue tracking dye migrates toward the anode Note that the dye front leaves the IPG strip 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 41 Table 13 Immobiline DryStrip IEF guidelines for Multiphor II Immobiline Cup loading or Rehydration loading Immobiline Cup loading DryStrip EPS 3501 XL power supply in gradient mode DryStrip TEPS 3501 XL power supply in gradient mode Length pH Phase Voltage Duration kVh Length pH Phase Voltage Duration kVh range s V h min range s V h min 7cm 4 7 1 200 0 01 0 001 7cm 6 11 1 200 0 01 0 01 2 3500 1 30 2 8 2 3 500 1 30 2 8 3 3 500 0 55 1 30 3 2 5 2 3 3 500 0 40 2 3 Total 2 25 3 00 6 8 Total 2 10 5 3 10 1 200 0 01 0 001 3 10NL 2 3500 1 30 2 8 3 3 500 0 35 1 05 2 2 3 Total 2 05 2 35 5 6 5 llem 4 7 1 300 0 01 0 001 llem 6 11 1 300 0 01 0 01 2 3500 1 30 2 9 2 3 500 1 30 2 9 3 3 500 2 20 3 30
53. a cell organism or biological fluid a process which requires stringently controlled steps of sample preparation 2 D electrophoresis image detection and analysis spot identification and database searches The core technology of proteomics is 2 D electrophoresis At present there is no other technique that is capable of simultaneously resolving thousands of proteins in one separation procedure The replacement of classical first dimension carrier ampholyte pH gradients with well defined immobilized pH gradients has resulted in higher resolution improved interlaboratory reproducibility higher protein loading capacity and an extended basic pH limit for 2 D electrophoresis With the increased protein capacity micropreparative 2 D electrophoresis has accelerated spot identification by mass spectrometry and Edman sequencing With immobilized gradients stable as high as pH 12 basic proteins can be separated routinely where previously they were lost due to cathodic drift of carrier ampholyte gradients or suffered from the limited reproducibility of NEPHGE The remarkable improvements in 2 D electrophoresis resulting from immobilized pH gradient gels together with convenient new instruments for IPG IEE will make critical contributions to advances in proteome analysis It is my pleasure to introduce this manual on 2 D electrophoresis It clearly describes the actual and technical basis of the current state of the art 2 D separations using immobili
54. ading There are two possible rehydration conditions 1 Passive rehydration No electric field is applied during rehydration 2 Rehydration under voltage In some cases rehydration under a low voltage 30 120 V facilitates the entry of high molecular weight proteins 70 B Optional Apply electrode pads During isoelectric focusing the transport of ions proteins and IPG buffer to the electrodes is accompanied by transport of water For large sample loads and narrow Immobiline DryStrip gels better results are obtained by applying damp paper pads between the IPG strip and each strip holder electrode just before IEF to adsorb excess water 1 Prepare electrode pads To apply 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 wv Important Electrode pads must be damp not wet 2 Position electrode pads Using forceps or tweezers lift one end of the rehydrated IPG strip Position an electrode pad over the electrode then lower the IPG strip back into place Repeat at the other end 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 IEE 1 Prepare sample Prepare the sample in a solution similar in composition to the rehydration solution used 2 Apply sample Fig 19 Pipette the s
55. ample into either or both of the lateral wells at either end of the strip holder Introduce the sample below the 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 Note The IPG strip backing is impermeable do not apply the sample to the back of the strip Replace cover on strip holder Table 15 Rehydration solution volume per Immobiline DryStrip e IPG strip length cm Total volume per strip pl 7cm 125 ul F 11 cm 200 ul 13 cm 250 ul 18cm 340 ul 24 cm 450 ul Including sample if applied Fig 19 Applying sample after gel rehydration 46 2 6 2 IPG strip rehydration Ettan IPGphor Cup Loading Strip Holder Several sample application methods are available when using Ettan IPGphor Cup Loading Strip Holder e Cup loading is recommended for sample volumes up to 100 pl and a maximum protein concentration of 100 pg protein 100 pl sample solution 100 pl is the volume of the cup Larger sample loads can lead to increased protein precipitation in the sample cup e Rehydration loading is preferred for large sample volumes greater than 100 pl and sample amounts e Paper bridge loading is selected for very large sample volumes and preparative electro phoresis and is particularly applicable when using basic pH intervals pH 6 9 and pH 6 11 Details of appropriate sample loads for silver and Coomassie staining using reh
56. and apply as a larger volume Program a low initial voltage and increase voltage gradually Prolong equilibration time Add 30 glycerol and 6 M urea to the SDS equilibration buffer Place application pieces at the end of the Strips during second dimension electro phoresis to absorb excess water Prepare fresh solutions Use 0 1 w v SDS Table 28 Troubleshooting 2 D results continued Symptom Vertical regions of poor focusing E e Pi Poor representation of higher molecular weight proteins continues on following page Possible cause Impurities in sample Impurities in rehydration solution components Bubble between IPG strip and top surface of second dimension gel Flatbed gel format Urea crystals on the surface of the IPG strip Flatbed gel format Bubbles under the IPG strip The IPG strip was not fully rehydrated Proteolysis of sample Insufficient equilibration Poor transfer of protein from PGstrip to second dimension gel Poor entry of sample protein during rehydration Remedy Modify sample preparation See section 1 4 Removal of contaminants that affect 2 D results Use only high quality reagents De ionize urea solutions Ensure that no bubbles are trapped between the IPG strip and the top surface of the second dimension gel Allow residual equilibration solution to drain from the IPG strip before placing the strip on t
57. are missing unclear or in the wrong position continues on following page Possible cause Sample is insufficient Insufficient sample entered the IPG strip due to poor sample solubilization Sample contains impurities that prevent focusing The pH gradient is wrongly oriented Flatbed gel format IPG strip is placed wrong side down on second dimension gel Detection method was not sensitive enough Failure of detection reagents Protein carbamylation Protein oxidation Table 14 page 43 Troubleshooting first dimension IEF Multiphor II and Immobiline Table 19 page 55 Troubleshooting first dimension IEF IPGphor Table 25 page 66 Troubleshooting vertical second dimension SDS PAGE Table 27 page 71 Troubleshooting second dimension SDS Multiphor II flatbed system Remedy Increase the amount of sample applied Increase the concentration of the solubilizing components in the sample solution See section 1 5 Composition of sample solution Increase the focusing time or modify the sample preparation method See Chapter Sample Preparation The pointed end of the Immobiline DryStrip is the acidic end and should point toward the anode Ensure that the IPG strip is placed gel side down plastic backing upward on the SDS second dimension gel Use another detection method e g silver staining instead of Coomassie blue staining Check expiry dates on staining sol
58. 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 about min 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 instruction for Multiphor Il Buffer Strip Positioner Lock the positioner in place by turning the grey locking cam until the positioner cannot be moved 5 Position the cathodic buffer strip Fig 31 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 If the buffer strip breaks piece it together on the gel Note 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 Carefully 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 s
59. cal 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 51 lt lt 6 52 of samples with differing salt concentrations A gradual increase in voltage is particularly advised for higher protein loads 100 pg or more per IPG strip Many factors affect the amount of time required for complete focusing and each specific set of conditions e g sample and rehydration solution composition IPG strip length and pH gradient requires an empirical determination for optimal results An approximate time for complete focusing is given in the example protocols provided in Table 17 Factors that increase the required focusing time include residual ions which must move to the ends of the IPG strips 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 IPG strip resulting in a lower final voltage when the system is limited by the maximum current setting Longer focusing times may therefore be required at IPG Buffer Pharmalyte concentrations higher than 0 5 For higher protein loads up to 1 mg or more the final focusing step of each protocol can be e
60. citelececenenecnsacbewenes 13 Multiphor Il flatbed system cisiciasccanesucstnesieecensdiniianeatednasivendainenaednadtasuasineneeiesitehaeeeedndudensanenawedesaceds 14 Vertical Systems ah sheen acre te fi dateians essa tudor nd west sehen ened tes dawned E E oieausancnee 14 Labor to TC WIGU sereia E E A EE L5 Chapter 1 Sample Pe WAl ALON siccis a aa Ea 17 1 0 Sample preparation general strategy cccccceccecececeeceeeeeeeeeeeeseeeeeeseeaeeeseseeenaeees 17 1 1 Methods Or Cel IMIS UO gui spssin an nan ace oa tccnrwatedSaaienude tar duacataauaaseninae sd SAAE S 18 1 1 1 Gentle lysis methodS wiiiec visiacnsdatecwiedsanaea aime ndscieaaheuveowiedealtiidsuua nid Wainesaveesisuiimshehaisidecehea ahaa adiuissianSiinadahaviaiinuick 19 1 1 2 More vigorous lysis methodS sijcnynsaxinmnewaniersnmeratesmmenmnanebanmnbansunsiosvanawisstonmasnseaexaneorwanseoniaoneatawenes 20 1 2 Protection against proteolysis ois csssceteshanens20ina beioueds von ibddntadous airi ieir iio anirai 21 1 3 Precipitation DOC CAINS ose ccanecan mia gsens acsamntdeegaenstenddguranmnieaennsagediurminnedetmerabdeaames 22 1 4 Removal of contaminants that affect 2 D results cccccececeeeeeteeeeeeeeeeeeeeeeeeeeaeaeaes 23 to COMPOSITION OT Sanrio be SO WI IOM wie viasiaieiese sdseswara sie e aae aaa aunties la haanihdsd ae 25 Chapter 2 First dimension Isoelectric Focusing IEF 0 2sscecssssseeecessseeeecesseeescesseeeeeseseesecens 27 2 0 First dimension isoelectric fO
61. ck electrode leads on the tray to the Multiphor II unit 7 Place the Immobiline DryStrip aligner Pour about 10 ml of DryStrip Cover Fluid into the Immobiline DryStrip tray Place the Immobiline DryStrip aligner 12 groove side up into the tray on top of the DryStrip Cover Fluid The presence of air bubbles between the strip positions under the aligner will not affect the experiment Avoid getting DryStrip Cover Fluid on top of the aligner at this point B Prepare electrode strips 1 Cut electrode strips to size Cut two IEF electrode strips to a length of 110 mm 2 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 tissue paper to remove excess water Important Electrode strips must be damp not wet Excess water may cause streaking Note Steps A and B above should be completed before proceeding C IEF with rehydration loading 1 Remove the rehydrated IPG strip from the Immobiline DryStrip Reswelling Tray To remove an IPG strip from the Reswelling Tray slide the tip of a pair of forceps along the sloped end of the slot and into the slight depression under the IPG strip Grab the end of the strip with the forceps and lift the strip out of the tray 37 38 2 Position the IPG strip in the Immobiline DryStrip aligner Fig 12 Immediately transfer the rehydrated IPG strips to adjacent
62. d 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 IPG strip more very acidic and basic proteins can be separated e IPG strips have a higher loading capacity for protein 64 e The sample can be introduced into the IPG strip during rehydration 65 66 e Precast Immobiline DryStrip gels are available from Amersham Biosciences These ready made dry IPG strips eliminate the need to handle toxic acrylamide monomers preparation time and effort are significantly reduced and reproducibility of the pH gradient is assured A 2 2 Immobilized pH gradient selection Ready made IPG strips Immobiline DryStrip gels are available from Amersham Biosciences with strip lengths of 7 11 13 18 and 24 cm Choose shorter strips for fast screening or when the most abundant proteins are of interest Use longer strips for maximal resolution and loading capacity Choosing the pH Gradient Use a pH interval of 3 10 for an overview of total protein distribution With a linear gradient pH 3 10 the estimation of protein s isoelectric point pl is relatively easy For increased resolution between pH 5 and 7 use 3 10 NL Non Linear to distribute the proteins more evenly over the gel This is especially helpful when analyzing complex samples like serum cerebrospinal fluid extracts from E coli and yeast Combine pH 3 7 and 6 11 or
63. daveusvemtaendinnns 85 K Agarose sealing solution 6a ci cumusasutenavaxtamurn avaraxasdeusabenndanuruvesanauentastemedhasmeasarencheusucdansecavesteneonsdsacus 86 PUI CNN oeann EE cnisleaetnnecadasdessucestuenssusedeesss vueeevatauineessdueverecesd 87 Optimized silver staining of Ettan DALT gels using PIUsSOne Silver Staining Kit ProteiM cccuisutiadsnaissamndcennisvennuwhdanaveduhindsuweanheaaainainbeanciuanudae 87 ROIOTONCOS sirsinn eiaa E AE ar Er EE 89 Additional reading and reference material c0 cccccssseecseseecesseeccenseecenseeeeneessenees 94 Recommended additional consumables cccceecceseeeeeeeeceeesseeeeeeeeanseeeesseeeenaes 94 Ordering information nnsonenonnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nenanem 95 Introduction Introduction to the manual This handbook is intended as a guideline for performing high resolution 2 D electrophoresis Depending on the sample type and the nature of the investigation the procedures may need to be adjusted or optimized The manual is divided into four chapters Chapter 1 provides guidelines for sample preparation Chapter 2 details procedures for performing the first dimension of 2 D electrophoresis Chapter 3 contains general directions for subsequent second dimension electrophoresis of immobilized pH gradient IPG strips Chapter 4 discusses visualization and analysis of the 2 D electrophoresis results The 2 D protocols described here
64. de buffer Ensuring that the valve on the separation unit is set to circulate add the entire contents 75 ml of the 100x anode buffer included in the Ettan DALT Buffer Kit into the tank Fill the tank to the 7 5 fill line with distilled or deionized water in this way washing the 100x anode solution from the buffer seal 3 Switch the separation unit on 4 Turn on the pump to mix set separation unit to desired temperature Note Avoid pouring the 100x anode buffer onto the tubing by spreading the tubing elements apart using one hand while pouring the solution with the other hand Fig 23 Note A temperature of 25 C is recommended for electrophoresis Fig 23 Avoid pouring the 100x anode buffer onto the tubing by spreading the tubing elements apart with one hand while pouring the solution with the other 3 3 2 Ettan DALT precast gels Ettan DALT Gel 12 5 is a precast polyacrylamide gel for the second dimension of two dimensional electrophoresis The gel is cast onto a plastic support film The exact gel size is 255 x 196 x 1 mm The gel is a homogeneous 12 5 polyacrylamide gel cross linked with bisacrylamide It is intended to be used in the Ettan DALT twelve system together with the Ettan 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 reusable ca
65. de of the second dimension gel Often there are abundant proteins in the sample for which the pI and molecular weight are known These proteins can serve as internal standards Note The pI of a protein can depend on its chemical environment and thus can differ depending on the experimental conditions used Although marker proteins for pI estimation are available pI estimates based on their use are not necessarily valid 75 76 4 4 Further analysis of protein spots 4 4 1 Picking the spots The Ettan Spot picker is a robotic system that automatically picks selected protein spots from stained or destained gels using a pick list from the image analysis and transfers them into microplates Ettan DALT or ExcelGel precast gels on film supports or lab cast gels on glass or plastic films are stained with Coomassie silver or fluorescent dyes and two visible reference markers are pasted on each gel The gels are scanned using ImageScanner or Typhoon and analyzed using ImageMaster 2D Elite or Ettan Progenesis software The positions of selected protein spots are exported as a pick list to the Ettan Spot Picker The gels are placed into the instrument under liquid the camera detects the reference markers Control software converts spot pixel co ordinates into picking co ordinates and the Ettan Spot Picker selects and tranfers gel plugs into 96 well microplates 4 4 2 Digestion of the proteins The gel plugs are automatically digested in the Etta
66. e a sensitivity in between colloidal Coomassie and modified PlusOne Silver Staining Kit Protein staining 77 These techniques require fluorescence scanners but they are compatible with mass spectrometry and show a wide dynamic range for quantification Preserving the gels The film supported Ettan DALT and ExcelGel 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 drier or in between two sheets of wet cellophane locked in Easy Breeze drying frames 4 1 Blotting Second dimension gels can be blotted onto a nitrocellulose or PVDF membrane for immunochemical detection of specific proteins or chemical microsequencing Note The plastic backing on Ettan DALT and ExcelGel precast gels is removed with the Film Remover prior to electrotransfer see Ordering Information 4 2 Evaluation In theory the analysis of up to 15 000 proteins should be possible in one gel in practice however 5 000 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 to detect the appearance of a few new spot
67. e buffer strip positioner helps to get optimal results good reproducibility is achieved because of standardized placement of IPG strips and buffer strips and a straight run because the gel surface is covered Important A flatbed second dimension system is not recommended if the first dimension has been run on a pH 6 11 IPG strip 1 Equilibrate the IPG strips During the preparation of the ExcelGel SDS gel equilibrate the IPG strips as described in section 3 2 2 2 Prepare the Multiphor Il Electrophoresis Unit Set the temperature on the MultiTemp III Thermostatic Circulator to 15 C Pipette 2 5 to 3 0 ml of kerosene 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 lefthand corner of the film identifies the 12 5 or 14 i e anodic end Note The gel is cast on 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 grid on the cooling plate Note Avoid trapping bubbles between the gel and the cooling plate Avoid getting DryStrip Cover Fluid or kerosene on the gel surface
68. e gel strip is not centered in the channel Remove the cup and the electrodes center the strip reposition the cup and check again e The strip may not have rehydrated properly Examine the strip closely for a thin region in which the cup may not be able to seal properly Be sure to use the proper volume of rehydration solution for a sufficient time to allow complete rehydration 4 Pipette up to 100 pl of sample into the sample cup 5 Place the cover over the strip holder E Paper bridge loading Very large sample volumes and protein amounts can be applied with paper bridges which are placed between the anodic or cathodic end of the IPG strip and the electrode pad Paper bridges or electrode pads are cut from a 1 mm thick CleanGel electrode strip see Ordering information Solution containing up to 5 mg protein can be loaded on an 18 cm long narrow pH range Immobiline DryStrip gel 68 A large sample volume requires that a large paper pad or arrowhead paper bridges be applied at the other end of the IPG strip to absorb excess water Figure 22 below shows the arrangement used when sample is applied to a paper bridge positioned between the anode and an 18 cm long IPG strip Paper bridges are cut from the 1 mm thick CleanGel electrode strips to a size of 8 x 45 mm and 8 x 35 mm to fit at the anodic and cathodic end of the Cup Loading Strip Holder The paper bridge is soaked with distilled water and blotted with a tissue paper to become damp n
69. e recently the non thiol reductant tributyl phosphine TBP at a concentration of 2 mM has been used as a reductant for 2 D samples 62 However due to the limited solubility and instability of TBP in solution a thiol reductant such as DTT should be used to maintain proteins in their reduced state through rehydration and first dimension IEF if TBP is employed as a reductant during sample preparation 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 base 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 dimension disturbances Bases or buffers should be diluted to 5 mM or lower prior to loading the sample onto first dimension IEF A sample 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 in 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 A widely used sample solution is given in Appendix I solution A
70. eagents Thiol reagents can be added at a later stage PMSF is very toxic AEBSF Aminoethyl benzylsulfony fluoride AEBSF is similar to PMSF in AEBSF induced modifications can or Pefabloc SC Serine Protease its inhibitory activity but is potentially alter the pl of a protein Inhibitor more soluble and less toxic Use at concentrations up to 4 mM 1 mM EDTA or 1 mM EGTA Generally used at 1 mM These compounds inhibit metalloproteases by chelating free metal ions required for activity Peptide protease inhibitors e g leupeptin pepstatin aprotinin Leupeptin inhibits many serine Peptide protease inhibitors are bestatin and cysteine proteases e expensive e reversible inhibitors Pepstatin inhibits asparty e small peptides and thus may appear on e active in the presence of DTT proteases e g acidic the 2 D map depending on the size range e active at low concentrations under proteases such as pepsin separated by the second dimension gel a variety of conditions Aprotinin inhibits many Pepstatin does not inhibit any proteases that Use at 2 20 pg ml serine proteases are active at pH 9 Bestatin inhibits aminopeptidases TLCK TPCK Tosyl lysine chloromethyl ketone These similar compounds tosyl phenylalanine chloromethyl irreversibly inhibit many serine ketone and cysteine proteases Use at 0 1 0 5 mM Benzamidine Use at 1 3 mM Benzamidine inhibits serine proteases 21 1 3 Precipitation procedures Protei
71. ect the strip holder s corresponding to the IPG strip length chosen for the experiment Important Handle the ceramic strip holders with care as they are fragile Very Important Wash each strip holder with detergent to remove residual protein Use a neutral pH detergent such as the Ettan IPGphor Strip Holder Cleaning Solution to remove residual protein from these strip holders Ettan IPGphor Strip Holder Cleaning Solution has been specifically formulated for removing protein deposits and will not damage the strip holder Ettan IPGphor Strip Holder Cleaning Solution can be ordered in 950 ml bottles from Amersham Biosciences See Ordering information 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 a few hours overnight in a solution of 2 5 Ettan IPGphor Strip Holder Cleaning Solution in water 1 First rinse off the strip holder A mild liquid soap may be used to remove any residual DryStrip cover fluid 2 Squeeze a few drops of Ettan IPGphor Strip Holder Cleaning Solution into the strip holder slot 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 comple
72. ed 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 1 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 Note 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 23 Single percentage gel recipes preparation of stock solutions is described in Appendix I solutions E F G and H Final Gel Concentration 5 7 5 10 12 5 15 Monomer solution solution E 16 7 ml 25 ml 33 0 Mil 41 7 ml 50 ml 4x resolving gel buffer Solution F 25 ml 25 ml 25 ml 25 ml 25 ml 10 SDS Solution G 1 ml 1 ml 1 ml 1 ml 1 ml Double distilled water 56 8 ml 48 5 ml 40 2 ml 31 8 ml 23 5 ml 10 ammonium persulfate Solution H 500 ul 500 ul 500 ul 500 ul 500 ul TEMED 33 ul 33 jil 33 yl 33 il 33 ul Total volume 100 ml 100 ml 100 ml 100 ml 100 ml Add after deaeration 65 66 Light solution Final Concentration Monomer solution solution E 4x resolving gel buffer Solution F 10 SDS Solution G Double distilled water 10 ammonium persulfate Solution H TEMED Total volume Heavy solution Final Concentration Monomer solution solution E 4x resolving gel buffer Solution F Sucrose 10 SDS So
73. eeceeeeseeeeeeseeseseeeesesaeseseaeseeaeseeaeaees 64 3 3 8 Iro bleshootiNg semtaniaia cidtiubissnn aula tedecbida unk eblas cehancoiaiitasadelembnterahanatietdaaseapoubasbhinlausia IRANEN 66 3A MUM DMOF H Tat Bed SYST nica cadnigannenevaatieadenamsavduainetabanhandieenceaimisdduncensemeeDaadaned d 68 3 4 1 EXCEIGE preparation s cisviiiniceriniriatinramicinnincriasieinnainmamiehicinunia lardamhhinmenetaniinnalarackiantannsa eke 68 3 4 2 Applying The equilibrated IPG strip sancysivonaislcusicsanevsteaney ceunsilateaa enn stmt anesahinw diesen wen catneencuaneanes 69 3 4 3 Electrophoresis conditions ate wncenitnticndowntstuuvennsenadienetannsnnntucanswedsvidaonmiaucunasdacadannatnancenasenndenpiewns 70 SAA FOUN MOONS arrene e E T E sae E E E E E EEEE E S 71 Chapter 4 Visualization and evaluation of results cccccssssseeceesceesseeeeeesceesseeeeeeseaeneeeeesenes 73 AO Visualization of VESUILS osciiiirsnnierei neniani Aaea aai 73 E E OT ING PIPEN AE AAE EEIE E AE A ee 74 ME E E EAE A EEE A A EEN E EE N EEE A 74 43 SAC NL Ze OM of PS LES eerie es ap ea tania civil coche Ribose 75 4 4 Further analysis Of protein Spots vatieanciusersrnaimnrvbawabeeennatavantinies aE ai 76 44 1 Picking the SOS arisen ene nn EEEE a EEEE EEEE EAE EOE EEE EROA 76 4 4 2 Digestion of the proteins cccccccsscsersceeesssensseeeescesenseveeebsssenscaeseneesenseenaseeussaeeeeesenessenenseensens 76 4 4 3 MALDI ToF mass spectrometry s ersnesnersnerrrerrrrrrrrr
74. efer SE 600 14 x 15 11 2 5 16 x 15 2 or 4 1 1 5 13 2 5 16 x 71 2x7 1 3 Ettan DALT Gel 12 5 26 x20 12 1 18 24 5 Multiple shorter IPG strips 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 1 Up to 8 mini format separations can be simultaneously achieved using the shorter 8 cm glass plates Multiphor II flatbed system This system provides excellent resolution and relatively rapid separations in a large format gel Precast ExcelGel products offer the convenience of ready to use gels and buffer strips The Multiphor II flatbed system Fig 3 page 10 offers convenience and versatility as it can be used for both first dimension IEF as well as second dimension SDS PAGE The protein loading capacity of an IPG strip can exceed the capacity of the thin horizontal second dimension gel so thicker vertical second dimension gels are preferred for micro preparative separations The Multiphor II system is not recommended for the second dimension if pH 6 11 IPG strips have been used for the first dimension separation 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 For rapid results the mini gel units Hoefer miniVE Fig 4 page 10 or SE 260 are recom mended The second dimension separation is
75. entered and covers the entire width of the second dimension gel Ensure that the thermostatic circulator is connected to the Multiphor II Electro phoresis Unit and functioning correctly Ensure that the expiration dates on the buffer strips and ExcelGel have not elapsed Ensure that the buffer strips are placed firmly on the gel with no air bubbles trapped beneath them E Ensure that the IPG strip is placed firmly on the gel with no air bubbles trapped underneath Stroke the plastic backing of the IPG strip gently with a pair of forceps to remove trapped bubbles Ensure that the electrodes are aligned over the center of the buffer strips before lowering the electrode holder 71 72 Chapter 4 Visualization and evaluation of results 4 0 Visualization of results Most detection methods used for SDS gels can be applied to second dimension gels The following features are desired e High sensitivy e Wide linear range for quantification e Compatibility with mass spectrometry e Low toxicity and environmentally safe e 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 protein To employ these techniques the sample must consist of protein radiolabelled in vivo using either
76. ents of the 2 D Immobiline DryStrip Kit include a tray and electrode holder anode and cathode electrodes an Immobiline DryStrip aligner a sample cup bar and sample cups Procedures A and B below should be completed before the IPG strips are removed from the Reswelling Tray A Prepare the Immobiline DryStrip Kit 1 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 2 Confirm electrical connections on Multiphor Il Check that the red bridging cable in the Multiphor II unit is connected 3 Establish cooling Set the temperature on MultiTemp III Thermostatic Circulator to 20 C Position the cooling plate on the Multiphor II unit and ensure that the surface is level 6 Position the Immobiline DryStrip tray Pipette approximately 3 to 4 ml of 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 The DryStrip Cover Fluid at this point serves as an electrical insulating fluid to ensure good thermal contact between the cooling plate and the tray Connect the red and bla
77. er or carrier ampholytes e Apply the sample in dilute solutions 60 100 ug protein per 100 ul For micropreparative applications rehydration loading is recommended zx Fig 14 Attachment of sample cups to the cup bar Fig 15 Applying sample into sample cups and pressing of sample cups against IPG strips 2 5 4 Paper bridge loading Fig 16 Higher sample volumes and protein amounts can be applied with paper bridges which are placed between the anodic or cathodic end of the IPG strip and the electrode strip A large sample volume requires a large paper pad applied at the other side to absorb 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 16 The rehydrated IPG strip is positioned directly on the glass bottom of the Immobiline DryStrip tray The arrowheaded paper to which 375 pl sample solution has 39 40 been added is then positioned at the anodic or the cathodic end of the IPG strip To hold the paper bridge and IPG strip in place press a sample cup positioned on the sample cup bar down on top of the arrowhead Solution containing up to 10 mg protein in 850 pl sample solution applied to a 15 x 50 paper bridge can be loaded on a 18 cm long narrow pH range IPG DryStrip under favorable conditions 68 The application point anodic or cathodic is of primary importance for
78. es will reduce protein contamination that can produce spurious spots or bands in 2 D patterns Clean all assemblies that will contact the gels or sample with 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 Always use the highest quality reagents and the purest water available Some of the chemicals used in the procedures acrylamide N N methylenebisacrylamide TEMED ammonium persulfate and SDS are extremely 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 lab The safety sheets should be reviewed prior to starting the procedures in the manual 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 15 16 Chapter 1 Sample Preparation 1 0 Sample preparation general strategy Appropriate sample preparation is absolutely essential for good 2 D results Due to the great diversity of protein sample types and origins only general guidelines for sample preparation are provided in this guide The optimal procedure must be determined empirically for each sample type Ideally the
79. ete details Check that both external electrode contacts on the underside of each strip holder make metal to metal contact with the platform Close the safety lid At least two of the three pressure pads under the safety lid must press gently against the cover of each strip holder to ensure contact between the electrodes and the electrode areas Begin IEE As isoelectric focusing proceeds the bromophenol blue tracking dye migrates toward the anode Note that the dye front leaves the IPG strip 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 Note it is possible that the programmed maximum voltage will not be reached with the shorter IPG strips or with samples with high conductivity Table 17 Guidelines for Ettan IPGphor with rehydration loading IEF for Immobiline Dry Strip pH 4 7 3 10 3 10 NL and narrow pH intervals Voltage step and hold mode 50 pA IPG strip 0 5 IPG buffer 20 C for both rehydration and IEF Rehydration time 12 h The total rehydration time can be adjusted somewhat for convenience but must be greater than 10 h Immobiline DryStrip Rehydration loading Length pH range s Step and Voltage Step d
80. etected background is corrected spot density is quantified and spots are matched between up to 100 gels The software can also detect and graphically display quantitative changes in spot patterns e ImageMaster 2D Database software adds a database search facility that searches and queries across experiments and images and analyzes experiments for quantitative pattern relationships e Ettan Progenesis is a high throughput fully automated 2 D imaging software for parameter free spot detection No manual spot editing is required resulting in maximum reproducibility of evaluation results Its automated parameter free warping drives spot matching between different gels providing a 3 D view of spots Batch processing of all spot detection background removal gel warping gel averaging and spot matching improves speed of routine analysis 4 3 Standardization of results The 2 D electrophoresis technique is often used comparatively and thus requires a reproducible method for determining relative spot positions Because precast Immobiline DryStrip gels are highly reproducible the pI of a particular protein can be estimated from its focusing position along a linear pH gradient IPG strip Detailed information on Immobiline DryStrip pH gradients are found in the Amersham Biosciences brochure Immobiline DryStrip visualization of pH gradients 18 1140 60 The second dimension can be calibrated using molecular weight marker proteins loaded to the si
81. ever pressure applied by the cover will ensure complete contact when the lid of the Ettan IPGphor unit is closed Fig 21 Placing a filter paper pad at the end of the gel strip C Rehydration loading Mix the sample with rehydration solution see page 48 A Immobiline DryStrip Reswelling Tray When the IPG Strips are rehydrated with the sample proteins continue directly with isoelectric focusing D Cup loading 1 Place the sample cup over the IPG strip Do not place the cup with the feet over an alignment protrusion the cup will not seat squarely and seal against the IPG strip However the sample cup can straddle these protrusions Press the cup down until fully seated against the bottom of the strip holder The feet on the sample cup stop the cup at a height above the floor of the strip holder sufficient to seal against the strip without cutting into it Make sure the cup and electrodes are positioned properly prior to loading the sample in the cup For basic IPG strips superior focusing patterns are generally obtained when the sample cup is placed as close to the anodic electrode as possible 2 Optional To confirm the sample cup has sealed pipette 100 ul of Immobiline DryStrip Cover Fluid into the cup Check the seal Remove the fluid prior to loading the sample 3 Three common causes of leakage include e A foot of the cup is resting on top of one of the protrusions Reposition the cup and recheck for leakage e Th
82. f a pH 3 10 NL gradient requires Vh in excess of 2 2 5 fold of that stated in Table 13 69 Salt and buffer ions in the sample can require an increase of the time for phase 2 in comparison to the values given in Table 13 particularly when cup loading is used High ion concentrations in the sample can also require an increase of the total Vh requirement as these ions have to be transported to the ends of the IPG strips Larger quantities of protein require more time to focus Note 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 section 5 0 page 77 Vv 2 5 6 Protocol examples Multiphor II The protocols in Table 13 are suitable for first dimension isoelectric focusing of protein samples in typical analytical quantities see Tables 10 and 11 pages 35 and 36 with IPG Buffer concentrations of 0 5 to 2 in the rehydration solution Optimal focusing time will vary with the nature of the sample the amount of protein and how the sample is applied For higher protein loads up to 1 mg or more the final focusing step of each protocol can be extended with an additional 20 of the total recommended Vh if necessary Note Sample application onto pH 6 11 and 6 9 IPG strips by rehydration loading is less likely to result in high quality 2 D results and should be avoided
83. fore raising the voltage to maximum too high include a prolonged low voltage phase in the IEF protocol to allow the ions to move to the ends of the IPG strip 2 6 Ettan IPGphor Isoelectric Focusing System With the IPGphor Isoelectric Focusing system both rehydration of the IPG strip and IEF occur in individual strip holders Different strip holder lengths are available for different IPG strip lengths The Ettan IPGphor Strip Holder is made of thermally conductive ceramics with built in platinum electrodes and a transparent lid IPG strip holders have a special surface treatment to minimize protein adsorption Because some cleaning agents can damage the surface clean the strip holders only with the Ettan IPGphor Strip Holder Cleaning Solution as directed The sample can be loaded by simply including it in the rehydration solution or loaded separately just prior to IEF into small lateral sample wells or alternatively into the loading cups of Cup Loading Strip Holder Ettan IPGphor Cup Loading Strip Holder can accommodate Immobiline DryStrip gels up to 24 cm length and is equipped with movable platinum electrode contacts Once sample is applied to the IPG strip and a Strip Holder is in place on the Ettan IPGphor unit platform the remaining steps are carried out automatically according to the chosen protocol Up to 12 strip holders can be supported 2 6 1 IPG strip rehydration Ettan IPGphor Strip Holder 1 Prepare the strip holder s Sel
84. h glass plates Scavenge any excess or residual thiol reducing agent with iodoacetamide before loading the IPG strips onto the second dimension gel Use IPG Buffer as carrier ampholyte mixture Reduce concentration if necessary Add DNase and RNase to hydrolyze nucleic acids Note The proteins DNase and RNase may appear on the 2 D map Make fresh SDS electrophoresis buffer Clean electrophoresis unit Appendix Solutions The acrylamide N N methylenebisacrylamide TEMED ammonium persulfate and SDS in this appendix are extremely hazardous You should have a manufacturer s safety data sheet MSDS detailing the properties and precautions for all chemicals in your lab The safety sheets should be reviewed prior to starting the procedures in this manual 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 A Lysis solution 8 M urea 4 CHAPS 2 Pharmalyte 3 10 Final concentration Amount Urea FW 60 06 8 M 19 2 g CHAPS 4 wiv 1 6g Pharmalyte 3 10 2 800 ul Double distilled H20 to 40 ml If necessary the concentration of urea can be increased to 9 or 9 8 M t Other detergents Triton X 100 NP 40 and other non ionic or zwitterionic detergents can be used instead of CHAPS Note Protease inhibitors may be added if necessary B Rehydration stock solution without IPG
85. he dye front is 1 mm from the bottom of the gel t Currents up to 50 higher may be used if only two gels per unit are being run no divider plates and the unit is being cooled with a thermostatic circulator 3 3 7 Preparing SDS slab gels vertical systems The acrylamide TEMED ammonium persulfate and SDS used in this protocol are extremely hazardous You should have a manufacturer s safety data sheet MSDS detailing the properties and precautions for all chemicals in your lab The safety sheets should be reviewed prior to starting the procedures in this manual 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 The instructions provided below for the preparation of vertical SDS polyacrylamide gels employ the tris glycine system of Laemmli 72 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 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 instruction
86. he integral of the volts applied over the time 27 28 IEF performed under denaturing conditions gives the highest resolution and the cleanest results Complete denaturation and solubilization is achieved with a mixture of urea and detergent ensuring that each protein is present in only one configuration and aggregation and intermolecular interaction is minimized Net Charge 3 2 Isoelectric point pl Fig 8 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 The original method for first dimension IEF depended on carrier ampholyte generated pH gradients in polyacrylamide gel rods in tubes 1 2 Carrier ampholytes are small soluble amphoteric molecules with a high buffering capacity near their pI Commercial carrier ampholyte mixtures are comprised of hundreds of individual polymeric species with pls spanning a specific pH range When a voltage is applied across a carrier ampholyte mixture the carrier ampholytes with the highest pI and the most negative charge move toward the anode and the carrier ampholytes with the lowest pI 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 Although this basic method has been used
87. he second dimension gel Ensure that the IPG strip is placed firmly on the gel with no air bubbles trapped under neath Stroke the plastic backing of the IPG strip gently with a pair of forceps to remove trapped bubbles Ensure that the IPG strips are rehydrated with a sufficient volume of rehydration solution Remove any large bubbles trapped under the IPG strip after rehydration solution is applied Check that the rehydration solution is evenly Spread along the entire length of the IPG strip Prepare sample in a manner that limits proteolysis and or use protease inhibitors See section 1 2 Protection against proteolysis Prolong equilibration time Employ a low current sample entry phase in the second dimension electrophoresis run Use recommended volume of rehydration solution See Table 10 page 35 81 82 Table 28 Troubleshooting 2 D results continued Symptom Point streaking Background smear toward bottom of gel Background smear toward top of gel High background in top region of gel Possible cause Silver staining Dirty plates used to cast gel or particulate material on the surface of the gel DTT and other thiol reducing agents exacerbate this effect Silver or Coomassie blue staining Staining of carrier ampholytes Silver staining Nucleic acids in sample Protein contaminant in SDS electrophoresis buffer or dirty electrophoresis unit Remedy Properly was
88. hether 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 IEE These always include urea and one or more detergents Complete denaturation ensures that each protein is present in only one configu ration and that aggregation and intermolecular interaction is avoided The lysis solution solution A see Appendix I page 83 which contains urea and the zwitterionic detergent CHAPS has been found to be effective for solubilizing a wide range of samples Reductant and IPG Buffer are also frequently added to the sample solution to enhance sample solubility 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 electro phoresis It is always included in the 2 D sample solution at a concentration of at least 8 M Urea solubilizes and unfolds 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 54 56 A non ionic or zwitterionic detergent is always included in the sample solution to ensure complete sample solubilization and to prevent aggregation through hydrophobic interaction
89. horesis of proteins J Biol Chem 250 4007 4021 1975 Klose J Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues A novel approach to testing for induced point mutation in mammals Humangenetik 26 231 243 1975 Bjellqvist B Ek K Righetti P G Gianazza E G rg A Westermeier R Postel W Isoelectric focusing in immobilized pH gradients principle methodology and some applications J Biochem Biophys Methods 6 317 339 1982 G rg A Postel W G nther S Weser J Improved horizontal two dimensional electrophoresis with hybrid isoelectric focusing in immobilized pH gradients in the first dimension and laying on transfer to the second dimension Electrophoresis 6 599 604 1985 Gorg A Postel W G nther S The current state of two dimensional electrophoresis with immobilized pH gradients Electrophoresis 9 531 546 1988 Wilkins M R Pasquali C Appel R D Ou K Golaz O Sanchez J C Yan J X Gooley A A Hughes G Humphrey Smith I Williams K L Hochstrasser D F From proteins to proteomes Large scale protein identification by two dimensional electrophoresis and amino acid analysis BioTechnology 14 61 65 1996 Pennington S R Wilkins M R Hochstrasser D E Dunn M J Proteome analysis From protein characterization to biological function Trends in Cell Biology 7 168 173 1997 Gorg A Boguth G Obermaier C Posch A Weis
90. ial 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 19 20 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 5 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 Cool on ice between bursts Complete shearing is obtained when 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 in chilled French pressure from forcing suspension through a small with cell walls cell Apply pressure and collect extruded lysate orifice under high pressure bacteria algae yeasts Grinding 5 8 28 29 Some cell types can be opened by hand Solid tissues Tissue or cells are normally frozen with liquid grinding with a mortar and pestle microorganisms nitrogen and ground to a fine powder Alumina
91. ide Suitable sample load will vary greatly between samples and with the sensitivity of the staining method used 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 Fig 14 Place sample cups on the sample cup bar high enough on the bar to avoid touching the gel surface Position the sample cup bar so 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 towards the anode cathode until the spacer just touches the anodic cathodic electrode 4 Press the sample cups against the IPG strips Fig 14 Move the sample cups into position one sample cup above each IPG strip and press the sample cups down to ensure good contact with each IPG strip This is perhaps the most critical part of
92. ide flex the gel downward slightly and lower it slowly toward the glass plate from left to right 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 Removal of bubbles and excess buffer Use the roller 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 see Fig 25 After rolling the gel should adhere firmly to the glass 5 Close the cassette Snap the plastic frame cover to the glass plate see Fig 26 and press the edges tightly together 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 ait a 5 g al ae i h 7 F Fig 25 Pressing out air pockets between gel Fig 26 Closing the Precast Gel Cassette and glass plate 3 3 3 Equilibrate the IPG strip See section 3 2 2 for equilibration protocol 3 3 4 Applying the equilibrated IPG strip 1 Position the IPG strip Dip the equilibrated IPG strip from section 3 2 2 in the SDS electrophoresis buffer see Appendix solution J to lubricate it 61 62 Both types of Ettan DALT gel cassettes those for lab cast and for precast gels have a longer glass
93. in are performed using Amersham Biosciences products Equipment choices are discussed on page 12 and illustrated in Table 1 Introduction to two dimensional 2 D electrophoresis Two dimensional 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 sorts proteins according to two independent properties in two discrete steps the first dimension step isoelectric focusing IEF separates proteins according to their isoelectric points pI the second dimension step SDS polyacrylamide gel electrophoresis SDS PAGE separates proteins according to their molecular weights M relative molecular weight Each spot on the resulting two dimensional array corresponds to a single protein species in the sample Thousands of different proteins can thus be separated and information such as the protein pI the apparent molecular weight and the amount of each protein is obtained Two dimensional electrophoresis was first introduced by P H O Farrell 1 and J Klose 2 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 Back ground to IEF page 27 for more detail The power of 2 D electrophoresis as a biochemical separation technique has been recognized virtually since its introduction Its applica
94. in hundreds of 2 D electrophoresis studies it has several limitations that have prevented its more widespread application e Carrier ampholytes are mixed polymers that are not well characterized and suffer from batch to batch manufacturing variations These variations reduce the reproducibility of the first dimension separation e Carrier ampholyte pH gradients are unstable and have a tendency to drift usually toward the cathode over time Gradient drift adversely affects reproducibility by introducing a time variable Gradient drift also causes a flattening of the pH gradient at each end particularly above pH 9 rendering the 2 D technique less useful at pH extremes e The soft polyacrylamide tube gels have low mechanical stability The gel rods may stretch or break affecting reproducibility Results are often dependent on the skill of the operator As a result of the limitations and problems with carrier ampholyte pH gradients immobi lized pH gradients were developed and Amersham Biosciences s Immobiline chemicals were introduced for the generation of this type of pH gradient 3 Gorg et al 4 5 pioneered the development and use of IPG IFF for the first dimension of 2 D electrophoresis The techniques used today are largely based on the work of A Gorg and her colleagues An immobilized pH gradient IPG is created by covalently incorporating a gradient of acidic and basic buffering groups into a polyacrylamide gel at the time it is cast
95. ining The second equilibration with iodoacetamide is also used to minimize 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 electrophoresis 3 2 2 Equilibration steps Note The second dimension vertical gel must be ready for use prior to IPG strip equilibration See sections 3 3 and 3 4 for preparation of vertical and horizontal gels respectively 1 Prepare equilibration solution Prepare SDS Equilibration buffer see Appendix solution D This is a stock solution Just prior to use add 100 mg DTT per 10 ml SDS equilibration buffer 2 Equilibration Place the IPG strips in individual tubes with the support film toward the wall Add 10 ml of the DT T containing solution to each tube Cap the tube and place it on its side on a rocker Equilibrate for 15 min 3 Second equilibration A second equilibration may be performed with an iodoacetamide solution without DTT Prepare a solution of 250 mg iodoacetamide per 10 ml SDS equilibration buffer Note This second equilibration step reduces point streaking and other artifacts Add 10 ml of solution per tube Cap the tube place it on its side on a rocker and equilibrate for 15 min Equilibration of IPGstrips prior to SDS PAGE 2 SDS 50 mM Tris HCl pH 8 8 6 M urea 30 v v glycerol 0 002 bromophenol blue 15 min 10 ml 100 mg DIT 15 min 10 ml
96. ion possible to use varies greatly between sample types For larger sample loads rehydration loading is recommended Strip holder Ettan IPGphor Cup Loading Strip Holder is made of aluminum oxide ceramic for efficient heat transfer and temperature control during IEF Two areas of metal plating on the bottom extend up the sides of the strip holder These plated areas make contact with the power supply contact pads when placed on the Ettan IPGphor separation platform Ettan IPGphor can accomodate a maximum of 9 Cup Loading Strip Holders 47 48 Protrusions along the channel inside the strip holder align the rehydrated IPG strip keeping it straight and centered when placed inside the strip holder The anodic end of the strip holder is somewhat pointed to indicate the direction of placement of the pointed IPG strip The strip holders have a special surface treatment to minimize protein adsorption Because some cleaning agents can damage the surface clean the strip holders only with the Ettan IPG Strip Holder Cleaning Solution as directed The strip holders are very fragile and should be handled with care Electrodes The moveable electrode clips can be placed anywhere along the chamber where the electrode bosses make electrical contact with the conducting rails on the sides of the strip holder Strips from 7 cm to 24 cm long can be used Sample cups Sample cups can be placed almost anywhere along the length of the Ettan IPGphor C
97. ion 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 IPG strip The markers should contain 200 to 1 OOO ng of each component for Coomassie staining and about 10 to 50 ng of each component for silver staining 3 Seal the IPG strip in place For precast Ettan DALT gels the agarose sealing has two functions 1 Blockage of the narrow gap s between the gel edge s and the lateral spacer s to prevent leakage of the upper buffer 2 Preventing the IPG strip from moving or floating in the elecrophoresis buffer The second point is valid for all vertical systems Prepare agarose sealing solution for Ettan DALT precast gels using the agarose sealing solution from the Ettan DALT Buffer Kit If using the Laemmli buffer system see Appendix solution K Melt each aliquot as needed in a 100 C heat block each gel will require 1 to 1 5 ml It takes approximately 10 min to fully melt the agarose Tip An ideal time to carry out this step is during IPG strip equilibration Allow the agarose to cool until the tube can be held by fingers 60 C and then slowly pipette the amount required to seal the IPG strip in place Fig 29 Pipetting slowly avoids introducing bubbles Only apply the minimum quantity of agarose sealing solution required to cover the IPG strip Allow a minimum of 1 min for the agarose to cool and solidify
98. ips in a minimal volume of solution 1 Prepare the Reswelling Tray Fig 10 page 35 Slide the protective lid completely off the tray and level the tray by turning the leveling feet until the bubble in the Spirit level is centered Ensure the tray is clean and dry 2 Apply the rehydration solution Pipette the appropriate volume into each slot as indicated in Table 15 page 46 Typical composition of rehydration solution 8 M urea 0 5 w v CHAPS 0 2 w v DTT 0 5 v v IPG Buffer or Pharmalytes 0 002 bromophenol blue The solution is either mixed with the sample solution for rehydration loading or is applied as such for later cup loading or paper bridge loading Deliver the solution slowly at a central point in the slot Remove any larger bubbles Important To ensure complete fluid and sample uptake do not apply excess rehydration solution 3 Position the IPG strip Fig 11 page 35 Remove the protective cover from the IPG strip starting at the acidic pointed end because of its superior mechanical stability Position the IPG strip as shown in Figure 11 with the gel side down and the pointed end of the strip against the sloped end of the slot Lower the IPG strip onto the solution To help coat the entire IPG strip 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 IPG strip 4 Overlay the IPG strip with DryStrip Cover Fluid Overlay
99. isruption should be performed at cold temperatures 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 these methods is to be used see section 1 2 It is generally preferable to disrupt the sample material directly into a strongly denaturing lysis solution in order 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 References 17 and 18 contain general information on tissue disruption and cell lysis 1 1 1 Gentle lysis methods These methods are generally employed when the sample of interest consists of easily lysed cells such as tissue culture cells blood cells 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 differential centrifugation Some times these techniques are combined e g osmotic lysis following enzymatic treatment freeze thaw in the presence of detergent Table 4 Gentle lysis methods Cell disruption method Application General procedure Osmotic lysis 19 This very gentle method is well suited for Blood cells Suspend ce
100. it R Berkelman T Harry R A Westbrook J A Wheeler C H Dunn M J 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 Castellanos Serra L Hardy E Guerra M Estevez E Mehl E Frank R W Understanding the mechanism of the zinc ion stains of biomacromolecules in electrophoreseis gels Generalization of the reverse staining technique Electrophoresis 19 2398 2406 1998 nl M Morgan M E Minden J S Difference gel electrophoresis A single gel method for detecting changes in protein extracts Electrophoresis 18 2071 2077 1997 Steinberg T H Hangland R P Singer V I Applications of SYPRO Orange and SYPRO Red protein gel stains Anal Biochem 239 238 245 1996 Steinberg T H Lauber W M Berggren K Kemper C Yue S Patton W 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 Dunn M J eds pp 65 86 Bios Scientific Publishers Ltd 2001 Yan J X Harry R A Spilbey C Dunn M J Postelectrophoretic staining of proteins separated
101. l concentration Amount SDS Electrophoresis buffer see solution J 100 ml Agarose NA or M 0 5 0 5g Bromophenol blue 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 a heating stirrer until the agarose is completely dissolved Do not allow the solution to boil over Dispense 2 ml aliquots into screw cap tubes and store at room temperature Appendix II Optimized silver staining of Ettan DALT 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 following 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 pl per 250 ml rather than 50 pl per 200 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 Glutardialdehyde 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 5x 8 min Silver reaction Silver nitrate solution 2 5 w v 25 ml 60 min Formaldehyde 37 w v 0 1 ml
102. lls in a hypoosmotic 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 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 e Dilute the lysed sample into a solution containing an excess of non ionic or zwitterionic detergent OR e Separate the SDS from the sample protein by acetone precipitation See Table 7 and Table 8 section 1 3 and section 1 5 for details Enzymatic lysis 23 24 Cells with cell walls can be lysed gently Plant tissue Treat cells with enzyme in isoosmotic solution following enzymatic removal of the cell wall bacter
103. lowing rehydration immediately prior to IEE e g if proteolysis or other protein modifications are a concern overnight rehydration with sample may not be desired Better results are obtained on pH 6 11 or 6 9 IPG strips when the sample is loaded anodically in a sample cup 31 32 The following two diagrams represent a general rule of how to select the appropriate mode of sample application Multiphor Il system Analytical Preparative pH gradient 3 5 4 5 4 0 5 0 4 5 5 5 5 0 6 0 5 5 6 7 cup a7 9 7 loading 3 10 3 10 NL 6 9 6 l Guidelines for sample application after rehydration using the Multiphor II and Immobiline DryStrip Kit system are given on page 34 For cup loading sample is pipetted into sample cups precisely positioned on the surface of the IPG gels Up to 100 pl per strip can be applied through the sample cups up to 850 nl with paper bridge loading 68 Ettan IPGphor Isoelectric Focusing System Analytical Preparative pH gradient STP HON Sepider Sep Hole 35 45 4 0 5 0 4 5 5 5 5 0 6 0 5 5 6 7 on 4 7 3 7 loading 3 10 3 10 NL 6 9 6 l Ettan IPGphor system guidelines for sample application after rehydration are given in section 2 6 3 page 51 Sample is pipetted into sample application wells located at each end of the Strip Holder Up to 7 5 pl of sample solu
104. lusOne equilibration chemicals Tris Urea Glycerol 87 SDS Dithiothreitol DTT Bromophenol Blue Enzymes Deoxyribonuclease DNase Ribonuclease RNase A and RNase B Ribonuclease A RNase A Molecular Weight Markers M range 2 512 16 949 M range 14 400 94 000 IEF sample application pieces pl Calibration Kits Broad pl Kit pH 3 5 9 3 Low pl Kit pH 2 5 6 5 High pl kit pH 5 10 5 Carbamylyte Calibration Kit Automated and Multiple Gel Staining Silver Staining Kit Protein Hoefer Processor Plus Base Unit PTFE coated stainless steel tray 19x29 cm Accepts gels up to 16x26 cm Quantity 250g 1kg 250g 1 kg 11 11 25g 11 10g 10g 500 g 25g 25 ml 500 g 500 g 11 100 g lg 10g 20 mg lg 100 mg 200 pk Code No 17 1302 01 17 1302 02 17 1300 01 17 1300 02 17 1301 01 17 1303 01 17 1304 01 17 1306 01 17 0422 01 17 0554 01 17 1323 01 17 1311 01 17 1312 01 17 1321 01 17 1319 01 17 1325 01 17 1313 01 17 1318 01 17 1329 01 27 0516 01 27 0330 02 27 0323 01 80 1129 83 17 0446 01 80 1129 46 17 0471 01 17 0472 01 17 0473 01 17 0582 01 17 1150 01 80 6444 04 80 6444 80 99 100 Product Quantity Automated and Multiple Gel Staining continued PTFE coated stainless steel tray 29x35 cm Accepts gels up to 28x26 cm Blot Processing Tray Pack Protocol Guide Hoefer Automated Gel Stainer Staining Tray Set Coomassie tablets PhastGel Blue R 350 SYPRO
105. lution G Double distilled water 10 ammonium persulfate Solution H TEMED Total volume 5 7 5 8 4 ml 12 5 ml 12 5 ml 12 5 ml 500 ul 500 ul 28 5 ml 24 5 ml 170 ul 170 ul 17 ul 17 ul 50 ml 50 ml 10 12 5 16 7 ml 21 0 ml 12 5 ml 12 5 ml 7 5g 1 32 500 ul 500 ul 16 2 ml 11 7 mil 165 ul 165 pl 16 5 ul 16 5 ul 50 ml 50 ml Table 24 Recipes for gradient gels preparation of stock solutions is described in Appendix I solutions E F G and H 10 12 5 15 16 5 ml 21 0 ml 25 ml 12 5 ml 12 5 ml 12 5 ml 500 ul 500 ul 500 ul 20 0 ml 16 0 ml 12 0 ml 170 ul 170 ul 170 ul 17 ul 17 ul 17 ul 50 ml 50 ml 50 ml 15 17 5 20 25 0 ml 29 2 ml 33 3 ml 12 5 ml 12 5 ml 12 5 ml 7 5g 7 5g 7 5g 500 ul 500 ul 500 ul 7 7 ml 3 5 ml O ml 165 ul 165 ul 165 ul 16 5 ul 16 5 ul 16 5 ul 50 ml 50 ml 50 ml Add after deaeration 3 3 8 Troubleshooting Table 25 lists possible problems that could be encountered during vertical SDS PAGE and how to solve them Table 25 Troubleshooting vertical second dimension SDS PAGE Symptom No current at start of run Second dimension separation proceeds too slowly Dye front curves up smiles at the edges Dye front curves down frowns Possible cause Insufficient volume of buffer in upper or lower reservoir SDS electrophoresis buffer prepared incorrectly or resolving gel buffer _ prepared incorrectly Acrylamide solution is too old Gel is not properly cooled Cur
106. m acetate in methanol and then with acetone Residual acetone is evaporated 42 43 47 53 Limitations 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 4 on removal of salts 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 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 The method is complicated and time consuming 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 Precipitation and resuspension should be avoided if the aim of a 2 D experiment is complete and accurate representation of all the proteins in a sample Table 7 lists some of the precipitation techniques used If sample preparation requires precipitation typically only one precipitation technique is employed
107. n Digester the supernatant peptides are mixed with MALDI matrix material and spotted onto MALDI slides using Ettan Spotter This spot handling procedure can either be performed fully automatically in the integrated Ettan Spot Handling Workstation or semi automatically by manual transfer of gels and microplates between these instruments as stand alone units 4 4 3 MALDI ToF mass spectrometry In the Ettan MALDI ToF mass spectrometer a laser beam is fired into the dried peptide matrix spots for ionization of the peptides After accurate determination of the peptide masses databases are searched for identification of the original proteins Ettan MALDI ToF utilizes an advanced quadratic field reflectron Z reflectron for automatic protein identification by peptide mass fingerprinting The Z reflectron also allows de novo sequencing of peptides by post source decay Chapter 5 Troubleshooting 5 0 Troubleshooting 2 D results Table 28 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 Tables e Table 12 page 36 Troubleshooting IPG strip rehydration in Reswelling Tray DryStrip Kit Table 28 Troubleshooting 2 D results Symptom No distinct spots are visible Individual proteins appear as multiple spots or
108. n on the operation of any of the instruments described please also see the respective User Manual Selecting an IEF system Amersham Biosciences offers two different systems for the first dimension separation the Multiphor II system with associated accessories and the Ettan IPGphor Isoelectric Focusing System Multiphor II Fig 1 is a versatile system that can be used to perform several different electrophoresis techniques An advantage of the Multiphor II 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 without sample or including sample rehydration loading is performed in the Immobiline DryStrip Reswelling Tray After rehydration the IPG strips are transferred to the electrophoresis unit for first dimension IEF The electrophoresis system is comprised of the Multiphor II flatbed unit with Immobiline DryStrip Kit which also allows cup loading and paper bridge loading of the sample onto rehydrated IPG strips This system accommodates up to 12 rehydrated IPG strips of the same length for any one IEF protocol Power is supplied by the EPS 3501 XL power supply and temperature control is provided by the Multi Temp III Thermostatic Circulator The Ettan IPGphor Isoelectric Focusing System Fig 2 further simplifies the first dimension separation with a system dedicated to IEF separation on IPG strips The system is comprised of Ettan IPGphor S
109. n 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 Table 7 Precipitation procedures Precipitation method 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 TCA precipitation TCA trichloroacetic acid is a very effective protein precipitant Acetone precipitation This organic solvent is commonly used to precipitate proteins Many organic soluble contaminants e g detergents lipids will remain in solution 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 Precipitation with ammonium acetate in methanol following phenol extraction This technique has proven useful with plant samples containing high levels of interfering substances 22
110. n 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 76 73 74 Hoefer Processor Plus automates multistep staining processes for increased convenience and reproducibility Automated protocols were developed to use the 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 approx 5 ng per spot can be achieved 77 For complete details please refer to the Hoefer Processor Plus Protocol Guide The Staining Tray Set provides a convenient means of staining up to 4 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 soultion to drain rapidly Negative Zinc Imidazole staining has a detection limit of approx 15 ng protein spot 78 and is well compatible with mass spectrometry but it is a poor quantification technique Fluorescent labelling 79 and fluorescent staining with SYPRO dyes 80 83 hav
111. n 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 71 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 dithiothreitol DTT is also added to break any S S linkages 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 relation ship between the logarithm 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 The most commonly used buffer system for second dimension SDS PAGE is the tris glycine system described by Laemmli
112. ncentration of IPG Buffer Pharmalyte are e Improved sample solubilization e Increased tolerance to salt in sample e A more even conductivity in the gel The drawbacks of increased concentration of IPG Buffer Pharmalye are e Higher concentrations will limit the voltage use during IEF and increase the time required for the focusing step e Silver staining may require a prolonged fixing step to wash out carrier ampholyte that may cause staining background 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 multiple IPG strips 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 IPG strips See section 2 4 2 Tracking dye bromophenol blue allows IEF progress to be monitored at the beginning of the protocol If the tracking dye does not migrate toward the anode no current is flowing Note the dye leaves the strip well before the sample is focused Sample can be applied by including it in the rehydration solution Up to 1 mg of sample per strip can be diluted or dissolved in rehydration solution prior to IEE The amount of sample required is dictated in part by the detection or visualization method used Radiolabelling requires a very small amount of sample silver staining requires typically 1 to 100 pg of
113. nt current or constant power for the Ettan DALTtwelve in two steps During the initial migration and stacking period the current is approximately half of the value required for the separation Stop electrophoresis when the dye front is approximately 1 mm from the bottom of the gel For the smaller systems cooling is optional However temperature control improves gel to gel reproducibility especially if the ambient temperature of the laboratory fluctuates significantly Important Do not cool SDS gels below 10 C After electrophoresis remove gels from their gel cassettes in preparation for staining or blotting Notch or mark each gel at the upper corner nearest the pointed end of the IPG strip to identify the acidic end of the first dimension separation If IPG strips have been applied correctly onto the precast Ettan DALT gels this measure will not be necessary as the gels are cast on a support film 63 64 Table 20 Recommended electrophoresis conditions for second dimension vertical gels Step Current mA gel Power W gel Duration h min Hoefer miniVE or SE 260 1 5 mm thick gels 1 15 0 15 2 30 1 30 1 0 mm thick gels 1 10 0 15 2 20 1 30 Hoefer SE 600 1 5 mm thick gels 1 15 0 15 2 30t 5 00 1 0 mm thick gels 1 10 0 15 2 20t 5 00 Ettan DALTtwelve set temperature to 25 C 1 mm thick gels 1 2 5 0 30 lab cast and precast 2 17 max 180 4 30 The time shown is approximate Stop electrophoresis when t
114. obtaining good results Paper Dndpe Elechrode smp f i A f i imimotiline DOrysirip Gel Electrode Fig 16 Setup for sample application via a paper bridge 2 5 5 Isoelectric focusing guidelines IEF in the Multiphor II system is conducted at very high voltages up to 3 500 V and very low currents typically less than 1 mA due to the low ionic strength within IPG strips During IEF the current decreases 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 up to several hours With cup loading a low initial voltage minimizes sample aggregation and a low initial voltage generally allows the parallel separation of samples with differing salt concentrations The main factors determining the required Voltage hours Vh are the length of the IPG strips and the pH gradient used Sample composition rehydration solution composition and sample application mode influence the required Voltage hours Table 13 page 42 suggests Voltage hours suitable for most samples with rehydration loading or anodic cup loading Cathodic sample application on wide range gradients pH 3 10 requires considerably longer focusing times than those stated in Table 13 especially if SDS containing samples are used As an example a SDS solubilized serum protein sample applied at the cathodic end o
115. omberg A Blomberg L Norbeck J Fey S J Larsen P M Larsen M Roepstorff P Degand H Boutry M Posch A G rg A Interlaboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient two dimensional gel electrophoresis Electrophoresis 16 1935 1945 1995 Damerval C de Vienne D Zivy M Thiellement H Technical improvements in two dimensional electrophoresis increase the level of genetic variation detected in wheat seedling proteins Electrophoresis 7 52 54 1986 Wu E S Wang M Y Extraction of proteins for sodium dodecyl sulfate polyacrylamide gel electrophoresis from protease rich plant tissues Anal Biochem 139 100 103 1984 36 Ia OS 39 40 41 42 43 44 45 46 47 48 49 50 Sl a2 J 54 Harrison P A 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 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 Thiellement H de Vienne D Analysis of leaf proteins by two dimensional gel electrophoresis Plant Physiol 78 178 182 1985 Barret A J Salversen G Proteinase Inhibitors Elsevier Press Amsterdam 1986 North M J Pre
116. on of the non ionic detergent present must be at least 8 times higher than the concentration of any ionic detergent to ensure complete removal of SDS from the proteins Load less sample Micropreparative separations require clean sample Modify sample preparation to limit contaminants See section 1 4 Removal of contaminants that affect 2 D results Program a low initial voltage and increase voltage gradually Extend focusing time 79 Table 28 Troubleshooting 2 D results continued Symptom Horizontal streaking or incompletely focused spots continued Prominent vertical streak at the point of sample application when loading IPG strips using sample cups Vertical streaking continues on following page Possible cause Underfocusing Focusing time was not long enough to achieve Steady state focusing Overfocusing Extended focusing times over 100 000 Vh may result in electroendosmotic water and protein movement which can produce horizontal smearing Impurities in agarose overlay or equilibration solution Flatbed gel format Sample aggregation or precipitation Insufficient equilibration Flatbed gel format Electroendosmosis Second dimension buffer solutions prepared incorrectly Insufficient SDS in SDS electrophoresis buffer Remedy Prolong focusing time Reduce focusing time Prepare fresh agarose overlay and equilibration solution Dilute the sample
117. ot plugged into the power supply correctly It is normal for several hours to elapse before the sample dye leaves _ the sample cups The sample cups were pressed down so hard against the gel that they pushed through the gel to rest against the plastic backing This blocks the current and physically prevents the protein from entering the IPG strip continues on following page Remedy Sample cups are fragile and should not be used too many times Make sure the sample cups are aligned with the IPG strips Make sure the bottom of the sample cups are flat against the gel surface of the IPG strips Note Leaks can often be detected prior to sample application e Observe the IPG DryStrip Cover Fluid when it is poured into the Immobiline DryStrip Kit tray If it leaks in through the bottom of the Sample cups reposition the cups remove the cover fluid with a pipette and check for leakage again e 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 mportant the leaked detection dye must be removed from the sample cup before loading the sample Usually an IPG run starts close to 1 mA and drops into the uA range This depends on the number of IPG strips in the instrument Because the EPS 3501 XL can operate under very low currents it is recommended for use with Immobiline DryStrip Kit and Immobiline DryStrip gels Make sure the low c
118. ot wet Sample solution is applied to the paper bridge 450 ul for anodic sample application and 350 ul for cathodic sample application The rehydrated IPG strip is first positioned in the bottom of the stripholder Then the paper bridge positioned as indicated in Figure 22 With anodic application the anode is positioned as far out as possible in the electrode holder while the cathode is positioned close to the end of the IPG strip to ensure good contact between electrode pad and IPG strip A 6 mm soft plastic tubing is positioned as indicated in Figure 22 When the cover is placed over the strip holder it will press down the tubing and ensure good contact between the paper bridge and IPG strip Solutions containing up to 5 mg protein can be loaded on an 18 cm long narrow pH range IPG strip Note The application point anodic or cathodic is an important factor for obtaining good results Cows Angie w Cathode Paper bridge s i ia Cup Loading Strip Holder IFG strip Plastic tubing Fig 22 Equipment used for paper bridge loading of large sample volumes 2 6 3 Isoelectric focusing guidelines IEF in the Ettan IPGphor system is conducted at very high voltages up to 8 000 V and very low currents typically less than 50 pA per IPG strip due to the low ionic strength within IPG strips During IEF the current decreases while the voltage increases as proteins and other charged components migrate to their equilibrium positions A typi
119. otein nucleic acid interactions may also be helpful solubilize sample in SDS or at high pH Strongly denaturing conditions and detergents minimize protein lipid interactions Excess detergent may be necessary Precipitation with acetone removes some lipid Table 8 Contaminants that affect 2 D results continued Contaminant Reason for removal Removal techniques Phenolic compounds Phenolic compounds are present in many Prevent phenolic oxidation by employing plant tissues and can modify proteins reductants during tissue extraction through an enzyme catalyzed oxidative e g DTT 2 mercaptoethanol sulfite reaction 43 49 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 polyvinylpolypyrrolidone PVPP 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 to Insoluble material is particularly first dimension IEF problematic when the sample is applied using sample cups it can prevent protein entry into the IPG strip 1 5 Composition of sample solution In order to achieve a well focused first dimension separation sample proteins must be completely disaggregated and fully solubilized Regardless of w
120. plate The cassette should be laid on the bench with the longer glass plate down the protruding edge oriented towards the operator Place the strip with the acidic end to the left gel surface up onto the protruding edge of the longer glass plate see Fig 27 For other systems Position the IPG strip between the plates on the surface of the second dimension gel with the plastic backing against one of the glass plates Fig 27 With a thin plastic ruler gently push the IPG strip down so that the entire lower edge of the IPG strip is in contact with the top surface of the slab gel Fig 28 Ensure that no air bubbles are trapped between the IPG strip and the slab gel surfaces or between the gel backing and the glass plate Sidis emd Fig 27 Positioning an equilibrated IPG strip on the Fig 28 Pushing the IPG strip down to contact Precast Gel Cassette the gel slab 2 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 to 20 ul For less volume cut the sample application piece proportionally Place the IEF applicat
121. pnweninenian obra ed anwentoduiva en ca suxtnaied apadiweuwanticesxpasuvens eenvtasreued 40 2 5 6 Protocol examples Multiphor Il ccaatoneinonnexdxas dain ieeneneabiineaeavetaadana tens ueieekacautessdubsleeveaitiidasaean 41 2 5 7 Running a protocol st vesiesesestrenssirenscliscttdadonsstnanatdl dco ceeulahonntatednssiv vise micnod nee thinnest aidihdalaoud sive dutnisisce isla raul ainesh 41 2 5 8 Preservation of focused IPG SINS xiucusionnavapecstlzesacdpasnpannncsanenaweaps pdenpawisanchanancteupnespasdedxanexencees 43 20 9 MOU SMOCMING viereisiin e a ara aariate Noa aika iieiea in ei 43 2 6 Ettan IPGphor Isoelectric FOCUSING System ic ccscisnsnineawenaia sasvaunidausacauaecsnnncaneauwaan ada 44 2 6 1 IPG strip rehydration Ettan IPGphor Strip Holder cccccccceccec eee eeeeeeeeeeeaeeeseeeeeeeeeeaeeeeeeaeees 44 2 6 2 IPG strip rehydration Ettan IPGphor Cup Loading Strip Holder cccccecceceeeeeeeeeeeeeeaeeaeaeeees 47 2 6 3 Isoelectric focusing guidelines sctcscinniierisclacavsnianiaduiancierbiatinaidaaetdeuedasalscasennniueiaiersibenievcnbiesies 51 2 6 4 Protocol examples Ettan IPGphof inicstuntinatsasiessintaecenlubinnertnesneatihiaihabterbansnccannyeaupiaeoerenrameeiel 52 20 O RUNNIN d ProOtOCOl soere e a A E E oaeAi a NeaeEnNE 52 20 0 MOUDIESNOOUN E cerien EE E A E EEEE EEEE E AEREE AEE E E E EEE 55 Chapter 3 Second dimension SDS PAGE ccccccssssssseeeeescessseeeeseeeesseeeeseeeeesseeeeseaeenseeeeeses 57 3
122. process will result in the complete solubilization disaggregation denaturation and reduction of the proteins in the sample 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 complete 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 non specific 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 particula
123. r sample separations may be incomplete or distorted and information may be lost To fully analyze all intracellular proteins the cells must be effectively disrupted Choice of disruption method depends on whether the sample is from cells solid tissue or other biological material and whether the analysis is targeting all proteins or just a particular subcellular fraction Both gentle and vigorous lysis methods are discussed in section 1 1 Proteases may be liberated upon cell disruption Proteolysis greatly complicates analysis of the 2 D result thus the protein sample should be protected from proteolysis during cell disruption and subsequent preparation Protease inhibition is discussed in section 1 2 If only a subset of the proteins in a tissue or cell type is of interest prefractionation 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 prefractionated by solubility under different extraction conditions prior to 2 D electrophoresis References 9 10 11 and 12 describe examples of this approach See reference 13 for an overview of protein fractionation techniques 17 4 lt 8 Precipitation of the proteins in the sample and removal of inte
124. rder A Scheibe B Wildgruber R Weiss W The current state of two dimensional electrophoresis with immobilized pH gradients Electrophoresis 21 1037 1053 2000 Bjellqvist B Sanchez J C Pasquali C Ravier F Paquet N Frutiger S Hughes G J Hochstrasser D Micropreparative two dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins Electrophoresis 14 1375 1378 1993 Sanchez J C Rouge V Pisteur M Ravier F Tonella L Moosmayer M Wilkins M R Hochstrasser D F Improved and simplified in gel sample application using reswelling of dry immobilized pH gradients Electrophoresis 18 324 327 1997 Rabilloud T Valette C Lawrence J J 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 3 rd Ed WILEY VCH Weinheim 2001 Sabounchi Sch tt E Astr m J Olsson I Eklund A Grunewald J Bjellqvist B An Immobiline DryStrip application method enabling high capacity two dimensional gel electrophoresis Electrophoresis 21 3649 3656 2000 Bjellqvist B Pasquali Ch Ravier F Sanchez J Ch Hochstrasser D F A nonlinear wide range immobilized pH gradient for two dimensional electrophoresis and its definition in a relevant pH scale Electrophoresis 14
125. recipitation in the sample cup Maximum concentration of 100 ng protein 100 pl sample solution 100 pl is the volume of the cup is recommend This is a general recommendation which will function for most samples but the maximum concentration that is possible to use varies greatly between sample types For larger sample loads rehydration loading is recommended t Immobiline DryStrip narrow intervals pH 3 5 4 5 4 0 5 0 4 5 5 5 5 0 6 0 and 5 5 6 7 Table 12 Troubleshooting IPG strip rehydration in Reswelling Tray Symptom Uneven or incomplete Swelling of strips Possible cause Depending on the Immobline DryStrip pH interval and the pH of the reswelling solution either the basic end or the acidic end will Swell faster than the other The strip may not necessarly be of an even thickness following rehydration The unopened IPG strips package was stored at or above room temperature for too long IPG strips were stored at or above room temperature for too long Incorrect volume of rehydration solution used The rehydration time is too short Remedy Store IPG strips sealed at temperatures below 20 C Do not allow dry IPG strips to sit at room temperature for longer than 10 min Strips will pick up moisture from the air Make sure the correct amount of solution according to Table 10 is added to the slot in the Reswelling Tray Rehydrate the IPG strips for at least 10 h 2 5 2 Preparing for IEF The compon
126. red per vertical gel Casting system Volume ml Hoefer miniVE or SE 260 10 x 10 5 cm plates 1 mm thick spacers 10 1 5 mm thick spacers 15 Hoefer SE 600 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 4 Calculate the formulation of the gel solution The recipes given in Table 23 produce 100 ml of solution for a single percentage gel The recipes in Table 24 produce 50 ml each of light and heavy solution for a gradient gel These recipes are to be scaled up or down depending on the volume required 5 Prepare the gel solution Gel solution is prepared in a vacuum flask omitting the TEMED and ammonium persulfate Add a small magnetic stir bar Stopper the flask and apply a vacuum for several min while stirring on a magnetic stirrer Add the TEMED and ammonium persulfate and gently swirl the flask to mix being careful not to generate bubbles Immediately pour the gel 6 Pour and prepare the gel Fill the gel cassette to 3 to 10 mm below the top no stacking gel layer is required Overlay each gel with a layer of water saturated n or t butanol 3 ml immediately after pouring to minimize gel exposure to oxygen and to create a flat gel surface After allowing a minimum of 1 h for polymerization remove the overlay and rinse the gel surface with gel storage solution see Appendix solution 7 Storage of unus
127. rent or power too high Gel is poorly polymerized near _the spacers _ Improper instrument assembly SE 600 Leakage of upper reservoir continues on following page Remedy Ensure that both reservoirs contain enough SDS electrophoresis buffer to contact both upper and lower electrode wires Check for leaks Make fresh solutions Prepare fresh monomer stock solution During electrophoresis actively cool gel using a thermostatic circulator Use the maximum possible volume of buffer in the lower reservoir Limit current or power to values suggested in Table 20 Degas the gel solution or increase the amount _ of ammonium persulfate and TEMED by 50 _ Ensure that the gasket is not pinched Ensure that an adequate level of buffer is in the upper reservoir Table 25 Troubleshooting vertical second dimension SDS PAGE continued Symptom Second dimension separation proceeds slowly with high current Dye front is irregular Pronounced downward curving of the dye front on one side of the gel Distortion in the 2 D pattern Vertical gap in the 2 D pattern Possible cause All of the slots in the sealing assembly are not occupied by either gel cassettes or blank cassettes Anodic buffer has mixed with cathodic buffer from overfilling of either the cathodic reservoir or the anodic reservoir Poor uneven polymerization of gel The top surface of the second dimension gel is not flat
128. retic analysis Electrophoresis 17 813 829 1996 Rabilloud T Adessi C Giraudel A Lunardi J Improvement of the solubilization of proteins in two dimensional electrophoresis with immobilized pH gradients Electrophoresis 18 307 316 1997 89 90 17 18 19 20 Zs 22 23 24 23 26 27 28 2 30 31 EPA 33 34 i3 Bollag D M Edelstein S J Protein Methods Chapter 2 Protein Extraction Wiley Liss NY NY 1991 Scopes R K Protein purification Principles and practice 2nd Ed Chapter 2 Making an Extract Springer Verlag NY 1987 Dignam J D Preparation of extracts from higher eukaryotes Methods Enzymol 182 194 203 1990 Toda T Ishijima Y Matsushita H Yoshida M Kimura N 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 Appel R D Golaz O Pasquali C Ravier F Bairoch A Hochstrasser D F Inside SWISS 2DPAGE database Electrophoresis 16 1131 1151 1995 Portig I Pankuweit S Lottspeich E Maisch B Identification of stress proteins in endothelial cells Electrophoresis 17 803 808 1996 Cull M 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
129. rfering 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 3 Removal techniques which eliminate specific contaminants from the sample are described in section 1 4 as are the effects contaminants salts small ionic molecules ionic detergents nucleic acids polysaccharides lipids and phenolic compounds might have on the 2 D result if they are not removed In general it is advisable to keep sample preparation as simple as possible A sample with low protein concentrations and a high salt concentration for example could be diluted normally and analyzed or desalted then concentrated by lyophilization or precipitated with TCA and ice cold acetone and re solubilized with rehydration solution The first 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 removal steps may be necessary The composition of the sample solution is particularly critical for 2 D because solubilization treatments for the first dimension separation must not affect the protein pI nor leave the sample in a highly conductive solution In general concentrated urea as well as one or more
130. rgent Nucleic acids DNA RNA Polysaccharides Lipids continues on following page Reason for removal 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 Nucleic acids increase sample viscosity and cause background smears High molecular weight nucleic acids can clog gel 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 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 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 Removal techniques Dilute the SDS containing sample into
131. rip is against the glass plate on the second dimension gel Immediately after pouring the gel overlay the surface with water saturated butanol Degas the gel solution Polymerization can be accelerated by increasing by 50 the amount of ammonium persulfate and TEMED used Polymerization can be slowed by decreasing by 33 the amount of ammonium persufhate and TEMED used Ensure that there is no leakage during gel casting Allow ExcelGel to dry for about 5 min after removing plastic cover and before applying buffer strips and IPG strip Remove the IPG strip and application pieces from the second dimension gel when the bromophenol blue dye from has moved away from the IPG strip by 4 6 mm Ensure that no bubbles are trapped under the second dimension gel during placement on the cooling plate Take care that nothing is dropped or splashed onto the surface of the second dimension gel Table 28 Troubleshooting 2 D results continued Symptom Possible cause Horizontal streaking or incompletely focused spots Sample not completely solubilized prior to application Sample is poorly soluble in rehydration solution Interfering substances Non protein impurities in the Sample can interfere with IEF causing horizontal streaking in the final 2 D result particularly toward the acidic side of the gel lonic impurities in sample lonic detergent in sample High sample load continues on following page
132. rrriririnrrrnrirrrirrrrnrrsrrrrrerurirrrrrrnrrrrrrserrnree 76 Chapter 5 MOGI SO OUIN Scie senatcstancevunesseseneecoandateataviatewaistuctanseiseeuccieaneactassieaceatactccasaimeessatveass 77 6 0 Troubleshooting 2 D results sexsi dessenatandtnaasakivetionndutIudunasebs eadvibentiausacehescacestnunasells 7 Appendini EE 83 UON ae E A E oaetunieesedes 83 As SiS SOlUUON creeo EE E E E an colteatianieinns 83 B Rehydration stock solution without IPG Buffer sxccesisivacisxerinsdtarcanscteneracatsuapierapodatnmaisnanderepecteancaned s 83 C Rehydration stock solution with IPG Buffer enesnsesnersnennreranrernrernrrnrrrsrrrnrrnrererernrrsrrrrrerrrrrrren 84 D SDS equilibration buffer cesnnduaveceasngetnanpaunsasantounsdparnuuacimenenlencisuanstsuvcxavasenanensasausiocsesauaratenmecnssansns 84 E 30 T 2 6 C monomer stock solution ia snssiccnatiariasiaisranneiareiniornariondsrirsialarinnaianaceiaGanaen 84 F 4x resolving g l ON er s yeere eaa ie E mica REEE EEEE EEEO EEA ane vacate 85 A Nae Sa ise ei ct aes rs E E A E E E E A eee er 85 H 10 ammonium persulfate wtecayig ssa vartenatinubitie ts aitenic atievnuietentanincdieenantaaeslsmuennann sdnecat teenbiimnuintanatecianitanenns 85 Mal storage SOlUtION ste veciesapacactmecsinsecuotecienamndansinacimidttadwtantemotae ondasuan diosa polametewhean A EEEE NEES 85 J SDS electrophoresis buffer wis ainsesiccareeveissactsverieean sins vsichalihiad nresevaaiseradmiunled cea ved sesaldiwassvendicendane
133. s Originally either of two similar non ionic detergents NP 40 or Triton X 100 was used 1 2 Subsequent studies have demonstrated that the zwitterionic detergent CHAPS is often more effective 57 New zwitterionic detergents have been developed and reported to improve the solubility of membrane proteins 58 59 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 into 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 60 25 26 Reducing agents are frequently included in the sample 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 Dithioerythreitol DTE is similar to DIT and can also be used as a reducing agent Originally 2 mercaptoethanol was used as a reductant but higher concentrations of the reductant are required and inherent impurities may result in artifacts 61 Mor
134. s W 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 versus horizontal systems Electrophoresis 16 1079 1086 1995 Lenstra J A 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 Herbert B R Walsh B J Tyler M I Traini M Sanchez J C Hochstrasser D F Williams K L Gooley A A Extraction of membrane proteins by differential solubilization for separation using two dimensional gel electrophoresis Electrophoresis 19 837 844 1998 Ramsby M L Makowski G S and Khairallah E A Differential detergent fractionation of isolated hepatocytes Biochemical immunochemical and two dimensional gel electrophoresis characterization of cytoskeletal and noncytoskeletal compartments Electrophoresis 15 265 277 1994 Taylor R S Wu C C Hays L G Eng J K Yates J R I and Howell K E 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 Corbett J M 2 dimensional polyacrylamide gel electrophoresis Methods Enzymol 271 177 203 1996 Rabilloud T Solubilization of proteins for electropho
135. s centered Ensure the tray is clean and dry 2 Apply the rehydration solution Prepare the rehydration solution including sample for rehydration loading or without sample for cup application Pipette the appropriate volume of rehydration solution into each slot as indicated in Table 10 Deliver the solution Slowly at a central point in the slot Remove any larger bubbles Important To ensure complete fluid and sample uptake do not apply excess rehydration solution 3 Position the IPG strip Fig 11 Remove the protective cover from the IPG strip starting at the acidic pointed end Removal from the acidic pointed end prevents damage to the basic square end of the IPG strip which is generally softer Position the IPG strip as shown in Figure 11 with the gel side down and the pointed end of the strip against the sloped end of the slot Lower the IPG strip onto the solution To help coat the entire IPG strip 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 IPG strip Table 10 Rehydration solution volume per IPG Strip IPG strip length cm Total volume per strip pl 7 em 125 ul 11 cm 200 ul ae 13 cm 250 ul a 18 cm 340 ul Fig 10 Sliding the ater 1501 rca Including sample if applied Reswelling Tray om ia ne Ti u a A ni ta n F is ii re ls ely Tw im m aii s a Fig 11 Positioning of an IPG strip on
136. s on gradient preparation see the instruction manual for the relevant gel 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 gradient the appropriate percentage gel is selected according to the range of separation desired see Table 21 Table 21 Recommended acrylamide concentrations for protein separation Acrylamide in resolving gel Separation size range M x 10 Single percentage 5 36 200 7 5 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 Prepare the gel solution The total volume of solution needed depends on the gel size the gel thickness and the number of gels cast Table 22 gives volumes of gel solution required per gel for the various possible vertical gel formats 3 Select gel thickness for Hoefer SE 600 MiniVE or SE 260 electrophoresis systems 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 Table 22 Volumes requi
137. s 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 the gel patterns Amersham Biosciences ImageMaster 2D Elite Software and 2D Database Software as well as Ettan Progenesis 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 The ImageScanner desktop instrument captures optical information in the visible wave length range over a range from 0 to more than 3 4 O D in reflection or transmission mode It scans 20 x 20 cm in 40 s at 300 dpi e Typhoon 8600 and 9200 series variable mode imagers have two excitation sources for fluorescence imaging a green 532 nm and a red 633 nm laser Typhoon 9400 series imager has an additional blue laser with two excitation lines 457 nm and 488 nm Typhoon series imagers can be used for high performance 4 color automated fluorescence detection storage phosphor imaging and chemiluminescence Comprehensive information on fluorescence imaging can be found in the Amersham Biosciences handbook Fluorescence Imaging principles and methods 63 0035 28 e ImageMaster 2D Elite provides the essential tools for analyzing complex protein samples separated by 2 D electrophoresis Protein spots are automatically d
138. s rehydrated with a solution containing IPG Buffer of the corresponding pH gradient t During phase 2 the voltage will rise from the voltage set for phase 1 to 3 500 V The voltage will remain at 3 500 V throughout phase 3 1 Immobiline DryStrip narrow intervals pH 3 5 4 5 4 0 5 0 4 5 5 5 5 0 6 0 and 5 5 6 7 2 5 8 Preservation of focused IPG strips After IEF proceed to the second dimension separation immediately or store the IPG strips at 70 C 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 18 and 24 cm strips fit into the Equilibration Tubes available from Amersham Biosciences 2 5 9 Troubleshooting Table 14 lists possible problems that could be encountered during IEF and how to solve them Table 14 Troubleshooting first dimension IEF Multiphor II and Immobiline DryStrip Kit Symptom Sample cups leak Low current No current at start of run Sample dye does not move out of the sample cup Possible cause Incorrect handling and placement of sample cups This is normal for IPG gels The gels have very _ low conductivity Power supply cannot detect the low LA range current and shuts off IPG Buffer omitted from rehydration solution No electrode contact or lack of electrical continuity IPG strip is improperly _ rehydrated The high voltage lead from the electrophoresis unit is n
139. sample and Coomassie blue staining and preparative applications require larger sample amounts 2 4 2 Rehydration solution preparation Typical composition of rehydration solution without sample or for dilution with sample solution 8 M urea 0 5 w v CHAPS 0 2 w v DTT 0 5 v v IPG Buffer or Pharmalyte 0 002 bromophenol blue 1 Prepare the rehydration stock solution Recommended formulations are listed in Appendix solutions B C and D select the formulation appropriate to the experiment Note Stock solution can be stored in 2 5 ml aliquots at 20 C 2 Just prior to use slowly thaw a 2 5 ml aliquot of stock solution Add the appropriate amount of IPG Buffer or Pharmalyte if it is not already included in the rehydration stock solution refer to Table 9 3 Add 7 mg DTT and sample if rehydration loading is desired refer to Table 11 Note DTT and the sample must be added fresh just prior to use 2 5 Multiphor Il and Immobiline DryStrip Kit 2 5 1 IPG strip rehydration Immobiline DryStrip Reswelling Tray The Immobiline DryStrip Reswelling Tray has twelve independent reservoir slots that can each hold a single IPG strip up to 24 cm long Separate slots allow the rehydration of individual IPG strips in a minimal volume of solution 1 Prepare the Reswelling Tray Fig 10 Slide the protective lid completely off the tray and level the tray by turning the leveling feet until the bubble in the Spirit level i
140. se Remedy Current is too low or zero Electrical continuity is impeded Check the external electrode contacts The electrodes at the bottom of the strip holder one at each end must make 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 strip holder Check that the IPG strip is fully rehydrated along its entire length Electrical contact at the electrodes is reduced by incomplete rehydration Voltage too low or The Ettan IPGphor protocol settings are Check that the current limit is properly set does not reach the incorrect for the experiment Check that the actual number of strips on maximum set value the Ettan IPGphor platform equals the number of strips 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 advisable only if sample solubility is a problem Sparking or burning Current limit setting is too high Do not exceed the maximum recommended inthe strips aaa Setting of SOWA per IPG Strip The IPG strip is not fully rehydrated Ensure the IPG strips are rehydrated with a sufficient volume of rehydration solution Remove any large bubbles trapped under the IPG strip after placing on rehydration solution Check tha
141. sham Biosciences appropriate to the pH range of the IPG strip and 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 10 16 54 56 Detergent solubilizes hydrophobic proteins and minimizes protein aggregation The detergent must have zero net charge use only non ionic and zwitterionic detergents CHAPS Triton X 100 or NP 40 in the range of 0 5 to 4 are most commonly used Reductant cleaves disulfide bonds to allow proteins to unfold completely DTT or DTE 20 to 100 mM are commonly used 2 Mercaptoethanol is not recommended because higher concentrations are required and impurities may result in artifacts 61 Tributyl phosphine TBP is not recommended as reductant for IEF due to its low solubility and poor stability in rehydration solution Reductants should be added directly before use IPG Buffer or Pharmalyte carrier ampholyte mixtures improve 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
142. 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 sample pH interval as the Immobiline DryStrip to be rehydrated Use IPG Buffer 3 5 5 0 for Immobiline DryStrip 3 5 4 5 and 4 0 5 0 Use IPG Buffer 6 11 for Immobiline DryStrip 6 9 and 6 11 Pharmalyte 3 10 may be used for separations on Immobiline DryStrip pH 3 10 and 3 10 NL Pharmalyte 5 8 may be used for separations on Immobiline DryStrip pH 4 7 33 34 Table 9 lists the recommended final concentration of IPG Buffer Pharmalyte for the rehydration solution The recommended IPG Buffer Pharmalyte concentration for the IPGphor system is 0 5 but up to 2 can be added if sample solubilization remains a problem Table 9 Addition of IPG Buffer or Pharmalyte to rehydration solution IEF system Sample application Second dimension Recommended concentration mode s system Multiphor Il Cup rehydration and Vertical gels flatbed 2 IPG Buffer 50 ul per 2 5 ml paper bridge loading 0 5 IPG Buffer 12 5 ul per 2 5 ml Ettan IPGphor Strip Holder Rehydration and sample Vertical gels flatbed 0 5 IPG Buffer 12 5 ul per 2 5 ml well loading 0 5 IPG Buffer 12 5 ul per 2 5 ml Cup Loading Strip Cup and paper bridge Vertical gels flatbed 2 IPG Buffer 50 ul per 2 5 ml Holder loading 0 5 IPG Buffer 12 5 ul per 2 5 ml The advantages of increased co
143. solving gel buffer 1 5 M Tris HCl pH 8 8 1 l Final concentration Amount Tris base FW 121 1 1 5 M Double distilled H20 HCI FW 36 46 181 7g 750 ml adjust to pH 8 8 Double distilled H20 to 1 Filter solution through a 0 45 pm filter Store at 4 C G 10 SDS Final concentration Amount SDS FW 288 38 10 w v Double distilled H20 5 0g to 50 ml Filter solution through a 0 45 pm filter Store at room temperature H 10 ammonium persulfate Final concentration Amount O lg Ammonium persulfate FW 228 20 10 w v Double distilled H20 to 1 ml Fresh ammonium persulfate crackles when water is added If it does not replace it with fresh stock Prepare just prior to use I Gel storage solution 0 375 M Tris HCl pH 8 8 0 1 SDS 200 ml Final concentration 4x Resolving gel buffer see solution F above 1x Amount 50 ml 10 SDS see above 0 1 2 ml Double distilled H20 Store at 4 C J SDS electrophoresis buffer 25 mM Tris HCl pH 8 3 192 mM glycine 0 1 SDS 10 l Final concentration to 200 ml Amount 30 3 g 144 0 g Tris base FW 121 1 25 mM Glycine FW 75 07 192 mM SDS FW 288 38 0 1 w v 10 0 g to 10 Double distilled H20 Because the pH of this solution need not be checked it can be made up directly in large reagent bottles marked at 10 l Store at room temperature 85 86 K Agarose sealing solution Fina
144. ssette and run in a vertical mode in the Ettan DALTtwelve Separation Unit Inserting the Ettan DALT Gel 12 5 into Ettan DALT Precast Gel Cassette 1 Open the gel package Cut around the gel on two sides at about 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 an Ettan DALT Precast Gel Cassette Place It on the bench top with the hinge down see Fig 24 Apply 1 ml gel buffer onto the glass plate as a streak along the spacer on the right edge of the glass plate see Fig 24 Fig 24 An opened Ettan DALT Precast Gel Cassette showing plastic frame cover left and glass plate right 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 s
145. sult If the sample is rehydrated into the IPG Strip the salt concentration in the rehydration solution should be lower than 10 mM If the sample is applied in sample cups salt concentrations of up 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 Endogenous small ionic molecules are present in any cell lysate These substances are often negatively charged and can result in poor focusing towards the anode Removal techniques Desalting can be performed by e 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 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 losses see section 1 3 TCA acetone precipitation is particularly effective at removing this sort of contaminant Other desalting techniques may be applied see above 23 24 Table 8 Contaminants that affect 2 D results continued Contaminant lonic dete
146. t the entire IPG strip surface is wetted a The IPG strip dried during IEF Always apply DryStrip Cover Fluid to prevent dehydration of a rehydrated IPG strip 55 56 Chapter 3 Second dimension SDS PAGE 3 0 Second dimension SDS PAGE overview After IEF the second dimension separation can be performed on various flatbed or vertical systems depending on factors such as those discussed in Equipment Choices on page 12 SDS PAGE consists of four steps 1 Preparing the second dimension gel 2 equilibrating the IPG strip s in SDS buffer 3 placing the equilibrated IPG strip on the SDS gel and 4 electrophoresis In this guide the equilibration step is described first because it is a protocol common to both vertical and flatbed systems Gel preparation IPG strip 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 they apply to vertical systems and Multiphor II flatbed systems respectively Note however that the second dimension gel must be prepared before the equilibration step is started 3 1 Background to SDS PAGE SDS PAGE SDS polyacrylamide gel electrophoresis 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 i
147. te removal of sticky proteins Rinse completely with distilled or deionized water after cleaning Handle clean strip holders with gloves to avoid contamination Important Strip holders may be baked boiled or autoclaved DO NOT EXPOSE THEM TO STRONG ACIDS OR BASES INCLUDING ALKALINE DETERGENTS Note The strip holder must be completely dry before use 2 Apply the rehydration solution Fig 17 Pipette the appropriate volume of rehydration solution into each strip holder as indicated in Table 15 Deliver the solution slowly at a central point in the strip holder channel away from the sample application wells Remove any larger bubbles Typical composition of rehydration solution 8 M urea 0 5 w v CHAPS 0 2 w v DTT 0 5 v v IPG Buffer or Pharmalyte 0 002 bromophenol blue Important To ensure complete sample uptake do not exceed the recommended volume of rehydration solution see Table 15 3 Position the IPG strip Fig 18 Remove the protective cover foil from the IPG strip starting at the acidic pointed end Removal from the acidic pointed end prevents damage to the basic Square end of the IPG strip which is generally softer Position the IPG strip with the gel side down and the pointed anodic end of the strip directed toward the pointed end of the Strip holder Pointed end first lower the IPG strip onto the solution To help coat the entire strip gently lift and lower the strip and slide it back and forth along
148. the surface of the solution tilting the strip holder slightly as needed to assure complete and even wetting Finally lower the cathodic square end of the IPG strip 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 dye the gel Be careful not to trap air bubbles under the IPG strip 4 Apply DryStrip Cover Fluid Apply IPG Cover Fluid to minimize evaporation and urea crystallization Pipette the fluid dropwise into one end of the strip holder until one half of the IPG strip is covered Then pipette the fluid dropwise into the other end of the strip holder adding fluid until the entire IPG strip is covered 5 Place the cover on the strip holder Pressure blocks on the underside of the cover assure that the IPG strip maintains good contact with the electrodes as the gel swells 6 Allow the IPG strip to rehydrate Rehydration can proceed on the bench top or on the Ettan IPGphor unit 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 protocol This is especially convenient if temperature control during rehydration is a concern i Fig 17 Applying rehydration Fig 18a b Positioning the IPG strip solution into the strip holder 45 A Rehydration lo
149. tion however has become significant only in the last few years as a result of a number of developments e The introduction of immobilized pH gradients and Immobiline reagents 3 brought superior resolution and reproducibility to first dimension IEF Based on this concept A Gorg and colleagues 4 5 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 merits of this technique is presented in section 2 1 Background to IEF page 27 e New mass spectrometry techniques have been developed that allow rapid identification and characterization of very small quantities of peptides and proteins extracted from single 2 D spots PBS e More powerful less expensive computers and software are now available rendering thorough computerized evaluations of the highly complex 2 D patterns economically feasible e Data about entire genomes or substantial fractions thereof for a number of organisms are now available allowing rapid identification of the gene encoding a protein separated by 2 D electrophoresis e The World Wide Web provides simple direct access to spot pattern databases for the comparison of electrophoresis results and genome sequence databases for assignment of sequence information A large and growing application of 2 D electrophoresis is
150. tion can be added to each side i e 15 pl per well or 30 pl total if both sides of both wells are used Up to 100 ul per strip can be applied through the sample cups when Ettan IPGphor Cup Loading Strip Holder is employed Furthermore rehydration loading and cup loading can be combined for the application of larger volumes Paper bridge loading can be performed in the Ettan IPGphor Cup Loading Strip Holder as well Up to 500 pl can be applied using the paper bridge method 2 4 IPG strip rehydration solution IPG strips must be rehydrated prior to IEF The IPG strips are rehydrated in the Immobiline DryStrip Reswelling Tray if either Multiphor II system or the Ettan IPGphor Cup Loading Strip Holder are used for IEF Using the Ettan IPGphor and the Strip Holder the strips are rehydrated in these strip holders Rehydration solution which may or may not include the sample is applied to the reservoir slots of the Reswelling Tray or Ettan IPGphor Strip Holder then the IPG strips are soaked individually Rehydrated strips are 3 mm wide and approximately 0 5 mm thick Note Cup Loading Strip Holder cannot be used for rehydration 2 4 1 Components of the rehydration solution The choice of the most appropriate rehydration solution for the sample will depend on its specific protein solubility requirements but a typical solution generally contains urea non ionic or zwitterionic detergent dithiothreitol DTT Pharmalytes or IPG Buffer Amer
151. tiphor 3 500 V Immobiline DryStrip Reswelling Tray 2 72 h Immobiline DryStrip Kit EPS 3501 XL power supply MultiTemp Ill Thermostatic Circulator IPGphor 8 000 V Ettan IPGphor Strip Holders of desired length 2 36 h Cup Loading Strip Holder for 7 24 cm strips Reswelling Tray for 7 24 cm strips Optimal focusing time varies widely depending on the IPG strip length and pH range and the nature of the sample Similar separations can generally be performed at least two fold faster with the IPGphor system than with the Multiphor II system t Higher voltages are not recommended for safety reasons A graphic guide for the selection of sample application methods and strip holders for Multiphor II as well as for Ettan IPGphor can be found on page 32 Selecting a second dimension system The second dimension separation may be performed in a flatbed or vertical system Table 3 matches the appropriate second dimension system and gel size with IPG strip length Further considerations are discussed below For a more complete discussion of the relative merits of flatbed vs vertical second dimensions consult reference 8 13 14 Table 3 Selection of a second dimension electrophoresis system Approx gel size Number of gels Gelthickness IPG strip length Total separation time w x l cm mm cm h m Flatbed Multiphor Il 24 5 11 1 0 5 all 1 45 ExcelGel 24 5 x 18 3 20 Vertical Hoefer mini VE or SE 260 8x9 2 1 15 7 1 30 Ho
152. trip Holders that serve both as rehydration and IEF chambers and the IPGphor unit which includes an 8 000 V power supply and built in temperature control Programmable parameters include rehydration temperature and duration IEF temperature and maximum current and the duration and voltage pattern of multiple steps for one separation Up to 12 strip holders of the same length can be placed on the Ettan IPGphor platform for any one protocol Because rehydration loading and IEF are performed con secutively without user intervention they can be performed unattended overnight For gradients at the high and low end of the pH scale as well as for very high protein loads on narrow pH range gradient strips Ettan IPGphor Cup Loading Strip Holder is employed for running the IPG strips gel side up Cup Loading Strip Holder allows additional ways of loading the sample cup loading and paper bridge loading Fewer IPG strip manipulations result in less error strip mix up contamination air contact and urea crystallization Separations are faster because of the substantially higher voltage that can be applied and the better temperature dissipation of the ceramic material of both types of strip holders Table 2 shows the key operating differences between the Multiphor II system and the Ettan IPGphor Isoelectric Focusing System for first dimension IEF Table 2 IEF system selection Maximum voltage Additional equipment required Time required for IEF Mul
153. trip 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 The buffer strips should sit snugly within the slots 3 4 2 Applying the equilibrated IPG strip See section 3 2 2 for the equilibration protocol 1 Drain moisture from IPG strips flatbed second dimension only After equilibration place the IPG strips on filter paper moistened with deionized water To help drain the equilibration solution place the IPG strips so they rest on an edge IPG strips can be left in this position for up to 10 min without noticeably affecting the spot sharpness Alternatively the IPG strips can be gently blotted with moistened filter paper to remove excess equilibration buffer 2 Position the IPG strip s Fig 32 Once the equilibrated IPG strips from section 3 2 2 have drained for at least 3 min place the IPG strips using forceps gel side down on the ExcelGel through the slot at position 2 Fig 31 Positioning the cathodic buffer strip on Fig 32 Positioning equilibrated IPG strips on Multiphor II Multiphor II 69 70 3 Position sample application pieces Fig 33 Using forceps place one IEF sample application piece at the end of each IPG strip underneath the plastic tab formed by the overhanging gel support film at each end of the IPG strip Be sure the application pieces touch the ends of the IPG strip Note Application pieces absorb
154. up Loading Strip Holder that is not blocked by a protrusion For proper sealing of the cup to the gel the feet of the sample cup must all rest on the bottom of the channel Cover The cover is used to ensure that the electrodes stay in place and that they are in good contact with the IPG strip The cover also applies gentle pressure to assure that the strip holder makes good contact with the IPGphor separation platform and power supply contact pads Immobiline DryStrip Cover Fluid The Immobiline DryStrip Cover Fluid is required to ensure that the rehydrated Immobiline DryStrip gels do not dry out during electrophoresis Without cover fluid the strips will dry out urea crystallize and the sample will not focus properly Electrode pads Although electrode pads are not required to make electrical contact between the IPG strip and the electrode they can improve the quality of results particularly on narrow range Immobiline DryStrip gels The pads absorb excess water as well as proteins with pls that are outside the pH range of the IPG strip A Immobiline DryStrip Reswelling Tray IPG strips must be rehydrated prior to IEF The IPG strips are rehydrated in the Immobiline DryStrip Reswelling Tray if Ettan IPGphor Cup Loading Strip Holders are used for IEE Immobiline DryStrip Reswelling Tray has 12 independent reservoir slots that can each hold a single IPG strip up to 24 cm long Separate slots allow the rehydration of individual IPG str
155. uration Volt hours voltage mode V h min kVh 7 cm 3 10 1 Step and Hold 500 0 30 0 25 3 10 NL 2 Step and Hold 1 000 0 30 0 5 4 7 3 Step and Hold 50007 1 40 7 5 Total 2 40 8 0 11 cm 3 10 1 Step and Hold 500 1 00 0 5 4 7 2 Step and Hold 1 000 1 00 1 0 3 Step and Hold 8 000 1 50 12 5 Total 3 50 14 0 13 cm 3 10 1 Step and Hold 500 1 00 0 5 3 10 NL 2 Step and Hold 1 000 1 00 1 0 4 7 3 Step and Hold 8 000 2 00 14 5 Total 4 00 16 0 18 cm 3 10 1 Step and Hold 500 1 00 0 5 3 10 NL 2 Step and Hold 1 000 1 00 1 0 4 7 3 Step and Hold 8 000 4 00 30 5 Total 6 00 32 0 Narrow intervals 1 Step and Hold 500 1 00 0 5 2 Step and Hold 1 000 1 00 1 0 3 Step and Hold 8 000 7 30 58 5 Total 9 30 60 0 24 cm 3 10 1 Step and Hold 500 1 00 0 5 3 10 NL 2 Step and Hold 1 000 1 00 1 0 4 7 3 Step and Hold 8 000 8 20 62 5 3 7 Total 10 20 64 0 Narrow intervals 1 Step and Hold 500 1 00 0 5 2 Step and Hold 1 000 1 00 1 0 3 Step and Hold 8 000 10 30 94 5 Total 12 30 96 0 This voltage may not be reached within the suggested step duration t The sample entry phase step 1 and 2 should be extended for high protein loads or for convenience if the strips are to be run overnight Narrow intervals 3 5 4 5 4 0 5 0 4 5 5 5 5 0 6 0 and 5 5 6 7 After IEF proceed to the second dimension separation immediately or store the IPG strips at 70 C 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
156. urrent shut off has been bypassed see power Supply instructions IPG runs may start in a current range that is _ not detectable by the power supply Always include IPG buffer or Pharmalyte in the rehydration solution Check to make sure all Multiphor Il 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 the red or black circle Ensure that the bridging cable _ under the cooling plate is properly installed Ensure that the IPG strip is rehydrated along its entire length Ensure that the plugs on the high voltage leads fit securely into the output jacks on the power supply Use the appropriate adapter if necessary Replace IPG strip and re apply sample cup 43 44 Table 14 Troubleshooting first dimension IEF Multiphor II and Immobiline DryStrip Kit continued Symptom Possible cause Remedy Sample dye does The ionic strength of the Dilute the sample as much as possible or just prior not move out of sample is higher than the to loading dialyze the sample to remove salts the sample cup gel As a result the field strength in the sample zone is Inadequate to move the protein out of the sample zone at an appreciable rate Sparking or burning Conductivity of the Ensure the sample is adequately desalted of IPG strips sample IPG strips is Alternatively be
157. use Remedy Vertical streaking Incorrectly prepared equilibration solution Prepare equilibration solution according aaa EO nstruetions Poor transfer of protein from IPG strip to Employ a low power or current sample entry second dimension gel phase in the second dimension electrophoresis tur Prolong entry phase if necessary Insufficient equilibration Prolong equilibration time Spots are vertically IPG strip is not placed properly Ensure that the plastic backing of the IPG strip doubled or is against the glass plate of the second twinned dimension cassette Poor representation Incorrectly prepared equilibration solution Prepare equilibration solution according to of higher molecular instructions weight proteins a Poor transfer of protein from IPG strip to Employ a low power or current sample entry second dimension gel phase in the second dimension electrophoresis run Prolong entry phase if necessary 3 4 Multiphor Il flatbed system 3 4 1 ExcelGel preparation Two sizes of precast ExcelGel gradient SDS gels are recommended for 2 D electrophoresis the 110 x 250 mm gel homogeneous 12 5 and the 180 x 250 mm gel which contains a 12 14 acrylamide gradient Either gel accepts a single 24 18 or 13 cm IPG strip two 11 cm or three 7 cm IPG strips Placing shorter IPG strips end to end is ideal for compara tive studies For maximum resolution the larger gel coupled with the 24 cm or 18 cm IPG strip is the best choice Using th
158. utions Prepare fresh staining solutions Do not heat any solutions containing urea above 30 C as cyanate a urea degradation product will carbamylate proteins changing their pl DTT in the rehydration and equilibration solutions keeps the disulfide bonds reduced For additional protection include an iodoacet amide treatment during equilibration prior to the second dimension separation lodoacet amide alkylates the thiol groups to prevent the reduced proteins from reoxidizing ZI Table 28 Troubleshooting 2 D results continued Symptom Spots are vertically doubled or twinned pE e My 4 d n M l me Distortion of 2 D pattern ir da continues on following page 78 Possible cause Vertical gel format IPG strip is not placed properly Vertical gel format The top surface of the second dimension gel is not flat Vertical gel format Uneven polymerization of gel due to incomplete polymerization too rapid polymerization or leakage during gel casting Flatbed gel format Moisture on the surface of the second dimension gel Flatbed gel format IPG strip not removed during electrophoresis Flatbed gel format Air bubbles under the second dimension gel cause uneven migration due to poor heat transfer Flatbed gel format Water drops or pieces of buffer strip on the surface of the second dimension gel Remedy Ensure that the plastic backing of the IPG St
159. vention of unwanted proteolysis In Proteolytic Enzymes A Practical Approach Beynon R J Bond J S eds pp 105 124 IRL Press Oxford 1989 Salvesen G Nagase H Inhibition of proteolytic enzymes In Proteolytic Enzymes A Practical Approach Beynon R J Bond J S eds pp 83 104 IRL Press Oxford 1989 Hurkman W J 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 Seifter S Precipitation techniques Methods Enzymol 182 285 300 1990 Cremer E 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 Philip R Tan Y H 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 Grosset J Chartier Y Cleyet Marel J C 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 Arundel P High resolution two dimensional electrophoreisis of plant proteins Anal Biochem 172 8 15
160. water that flows out of the IPG strips during electrophoresis 4 Ensure contact between IPG strip and ExcelGel Make sure that the IPG strip is in full direct contact with the SDS gel To remove any bubbles stroke the plastic backing of the IPG strip gently with a spatula or forceps 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 IPG strip Pipette the markers onto the extra sample application piece Apply the markers in a volume of 15 to 20 ul For less volume cut the sample application piece proportionally The markers should contain 200 to 1 OOO ng of each component for Coomassie staining and about 10 to 50 ng of each component for silver staining 6 Position electrodes Fig 34 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 the electrode connectors and carefully lower the electrode holder onto the buffer strips Fig 33 Positioning of application pieces Fig 34 Positioning electrodes 3 4 3 Electrophoresis conditions Place the safety lid on the Multiphor II Connect the power supply Recommended electrical settings and running times are listed in Table 26 Table 26 Electrophoresis conditions for ExcelGel Step Voltage V Current mA Power W Duration h min ExcelGel SDS 12 5 1 120 20 30 0 40 Open
161. xtended with an additional 20 of the total recommended Vh if necessary Note Exceeding the current limit of 50 pA IPG strip is not recommended as this may result in excessive heat generation and may damage the IPG strip and or strip holder Under extreme circumstances the IPG strip may burn Note Over focusing can sometimes occur on longer runs and may contribute to horizontal streaking visible in the 2 D result See also section 5 0 page 77 2 6 4 Protocol examples Ettan IPGphor These protocols are suitable for first dimension isoelectric focusing of protein samples suspended in rehydration solution in typical analytical quantities 1 to 100 pg The protocols are optimized for a rehydration solution containing 0 5 IPG buffer or Pharmalyte The recommended current limit is 50 pA IPG strip 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 user manual for instructions on how to program a protocol 2 6 5 Running a protocol Ensure that the strip holders are properly positioned on the Ettan IPGphor platform Use the guidemarks 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 User Manual for compl
162. ydration loading and cup loading using Ettan IPGphor Cup Loading Strip Holder are given in Table 16 As mentioned earlier cup loading has been found to improve protein 2 D patterns particularly with basic IPG strips pH 6 9 and pH 6 11 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 Table 16 Suitable sample loads for silver and Coomassie staining using rehydration loading and cup loading Immobiline DryStrip pH Suitable sample load pg of protein Silver stain Coomassie stain 7cm 4 7 4 8 20 120 6 11 8 16 40 240 3 10 3 10 NL 2 4 10 60 11 cm 4 7 10 20 50 300 6 11 20 40 100 600 3 10 L 4 8 20 120 13 cm 4 7 15 30 75 450 6 11 30 60 150 900 3 10 3 10 NL 8 15 40 240 18 cm 4 7 30 60 150 900 6 11 6 9 narrow interval 60 120 300 1 500 3 10 3 10 NL 15 30 75 450 24 cm 4 7 3 7 45 90 200 1 300 6 9 narrow interval 80 170 400 2 000 3 10 3 10 NL 20 40 100 600 Immobiline DryStrip narrow intervals pH 3 5 4 5 4 0 5 0 4 5 5 5 5 0 6 0 and 5 5 6 7 When using cup loading an increased sample concentration will lead to an increased risk of protein precipitation in the sample cup Maximum concentration of 100 ng protein 100 pl sample solution 100 pl is the volume of the cup is recommended This is a general recommendation which will function for most samples but the maximum concentrat
163. zed pH gradients for the first dimension it provides detailed protocols for new and experienced users and it includes an extensive bibliography Finally there is the pictorial troubleshooting guide a bit like photos from the album of Murphy s law that you wouldn t dare include in an official publication but here they are for all to learn from Angelika Gorg Technical University of Munich August 1998 Contents OG UCT Y OW isc ect teres aa sentence a i Ea 7 WTR ONC ION to Tne Manual serris EA a A E A eA 7 Introduction to two dimensional 2 D electrophoresis cceccececeeeeceeeceeeeceseeeeeeeeaeees 7 Symbols and abbreviations used in this handbook vincasnwei eins can tveniesnneurinsgaviadnnnuneacnesaadatiaibnaldandasaienieataulebek 8 Choices for first dimension lEF wichinassnciiasecnincoanenmncines tekst overt ueutunadeetateneruanbndinue ketetubacdeimiaacinreeecenis 9 Choices for second dimension SDS PAGE ccccccccccceec ee eee eee eee e ee eee eee eee eee eee ea ee eeaeeesetaeea ea eeeeaeeeea eats 10 Choices for second dimension SDS PAGE cccccccccecsececeee cence eeeeeeseeseaeseeseseeeeeeeeeseseeeeeeeesesgeseeaneesa 11 Equipment C MONG CS orsoni ieda aaae anai 12 Selecting an IEF system 2 ctniswatitamnsaactdeinimatsteaiatemen bahamas ened ait iatamienideanihainesatadanneabicad lahat ima aduide tien 12 Selecting a second dimension system xwscuntwan esters ntwnscanee ever itneeianasedaesebinstorawass maatiebiai
164. zontally on a flatbed electrophoresis unit Advantages of using the flatbed format include the following e Isoelectric focusing requires efficient cooling for close temperature control which can be effectively achieved on a horizontal ceramic cooling plate connected to a thermostatic circulator or a Peltier cooling plate e JEF requires high field strengths to obtain sharply focused bands thus 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 The IPG strips are rehydrated in a solution containing the necessary additives and optionally the sample proteins Rehydration solution is described in detail in section 2 4 page 33 IEF is performed by gradually increasing the voltage across the IPG strips to at least 3 500 V and maintaining this voltage for at least several thousand Volt hours After IEF the IPG strips are equilibrated in equilibration solution and applied onto flatbed or vertical SDS polyacrylamide gels When IPG strips are used for the first dimension separation the resultant 2 D maps are superior in terms of resolution and reproducibility IPG strips are a marked improvement over tube gels with carrier ampholyte generated pH gradients e The first dimension separation is more reproducible because the covalently fixed gradient cannot drift e Plastic backed IPG strips are easy to handle They can be picked up at either en

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