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1. Place 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 Position the electrodes Figure 12 o A b Each electrode has a side marked red anode or black cathode Align each electrode over an electrode strip d ensuring that the marked side corre sponds 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 d X dA Figure 12 20 USING IMMOBILIZED PH GRADIENTS FIRST DIMENSION ISOELECTRIC FOCUSING D Optional Apply sample after gel rehydration If the sample was not applied by means of the rehydra tion 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 12 These values should be regarded as only a general guide Suit able sample load will vary greatly among samples and with the sensitivity of the staining method used TABLE 12 SUITABLE SAMPLE LOADS WITH SAMPLE CUPS Immobiline DryStrip Suitable sample load ug of protein lem pH4 7L 4 8 M 3 933 p TOS o aU E I A
2. 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 182 154 174 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 identifi cation of Brucella melitensis proteins by two dimensional electrophoresis and microsequencing Electrophoresis 18 156 162 1997 Ames G EL Nikaido K Two dimensional gel electrophoresis of membrane proteins Biochemistry 15 616 623 1976 25 26 27 28 29 30 31 32 33 34 35 36 REFERENCES G rg 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 Eleciropboresis 9 681 692 1988 Posch A van den Berg B M Burg H C J G rg A Genetic variability of carrot seed proteins analyzed by and two dimensional electrophoresis with immobilized pH gradients Electrophoresis 16 1312 1316 1995 Geigenheimer P Preparation of extra
3. ate fe grains Double distilled H O Store in 40 ml aliquots at 20 C This is a stock solution Prior to use DTT or lodoacetamide is added See section 4 2 2 to 200 ml E Monomer stock solution 30 acrylamide 0 8 N N methylenebisacrylamide 200 ml APPENDIX Final concentration Amount Acrylamide FW 71 08 oO ce MAE LJ N N methylenebisacrylamide EA i ENN CA Double distilled H 0 to 200 ml Filter solution through a 0 45 um filler Store at 4 C away from light F 4X Resolving gel buffer 1 5 M Tris Cl pH 8 8 1000 ml Final concentration Amount Wis base WAZ 18M TBS Double distilled ne 0m HON 388 o Abt to pH 88 Double distilled H 0 to 1000 ml Filter solution through a 0 45 um filter Store at 4 C G 10 SDS Final concentration Amount SDS PW 288 38 NEU LEE ua Double distilled H 0 to 50 ml Filter solution through a 0 45 um filter Store at room temperature H 10 Ammonium persulphate Final concentration Amount Ammonium persulphate FW 228 20 10 Mg ss Double distilled H 0 to 1 0 ml Fresh ammonium persulphate crackles when water is added If it does not replace it with fresh stock Prepare just prior to use l Gel storage solution 0 375 M Tris Cl pH 8 8 0 1 SDS 200 ml Final concentration Amount AX resolving gel buffer see solution P YO Om 1096 SDS see solution G g 0 1 2 ml g Double distilled H20 to 200 ml Store at 4 C J SD
4. Remove any large bubbles between the tray and the cooling plate small bubbles can be ignored The IPG Cover Fluid at this point serves to ensure good thermal contact between the cooling plate and the tray Note It is normal for the basic end to swell faster than the Remedy Do not allow dry IPG strips to sit at room temperature for longer than 10 minutes Strips will pick up moisture from the air Store IPG strips well sealed at temperatures below 20 C Make sure the correct amount of solution is added to the slot in the Reswelling Tray Rehydrate the IPG strips for at least 10 hours Connect the red and black electrode leads on the tray to the Multiphor II unit Q Place the DryStrip aligner Pour about 15 ml of IPG Cover Fluid into the Immobi line DryStrip tray Place the DryStrip aligner 13 groove side up into the tray on top of the IPG Cover Fluid The presence of air bubbles between the strip positions under the DryStrip aligner will not affect the experiment Avoid getting IPG Cover Fluid on top of the aligner at this point as it interferes with visualization of the grooves B Prepare electrode strips O Cut electrode strips to size Cut two IEF electrode strips to a length of 110 mm O Soak electrode strips with distilled water Place the electrode strips on a clean flat surface such as a glass plate Soak each electrode strip with 0 5 ml distilled water Blot with filter paper to remove ex
5. continued Choices for second dimension SDS PAGE Figure 6 Hoefer DALT Multiphor II flatbed system 24 5 x 11 cm or 24 5 x 18 cm gels Choice Factors Precast gels offered ExcelGel 8 18 24 5 x 11 cm ExcelGel XL 12 14 24 5 x 18 cm 1 Relatively rapid 4 hours or less for electrophoresis 1 High resolution 1 All available IPG strip lengths can be used Hoefer miniVE or SE 260 mini vertical 8 x 9 cm gels Choice Factors 1 Rapid 1 2 hours for electrophoresis 1 Best for 7 cm IPG strips Hoefer SE 600 standard vertical 14 or 16 x 15 cm gels Choice Factors a 4 5 hours for electrophoresis E Intermediate separation 15 cm gel length E Intermediate throughput up to four gels simultaneously 3 Best for 13 cm IPG strips Hoefer DALT large format vertical 24 x 19 cm gels Choice Factors 7 hours to overnight electrophoresis 1 Highest resolution 19 cm gel length 1 Highest possible protein capacity 1 High throughput up to 10 gels simultaneously 3 Best for 18 cm IPG strips 2 D ELECTROPHORESIS 3 INTRODUCTION 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 IEF First dimension IEF is performed on a flatbed system at very high volt ages with active temperature control Next strip equili bration in SDS con
6. Do not expose samples to repeated thawing 1 Remove all particulate material by ultracentrifugation Solid particles and lipids must be removed because they will block the gel pores 1 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 TABLE 4 GENTLE LYSIS METHODS Cell disruption method Osmotic lysis 16 This very gentle method is well suited for applications in which the lysate is to be subsequently fractionated into subcellular components SAMPLE Application Blood cells tissue culture cells PREPARATION cause urea to hydrolyze to isocyanate which modifies proteins by carbamylation For more specific guidance on preparing samples for application to IPG strips see 11 13 2 1 Methods of cell disruption Listed in Tables 4 and 5 are a few standard disruption methods both mechanical and chemical Cell disruption should be performed at cold temperatures Keep the sample on ice as much as possible and use chilled solu tions Proteases may be liberated upon cell disruption thus the protein sample should be protected from prote olysis if one of these methods is to be used See section 2 2 It is generally preferable to disrupt the sample mate rial directly into a strongly denaturing lysis solution in order to rapidly inactivate proteases and other enzymatic activities that may modify proteins Cell disrup
7. FIRST DIMENSION ISOELECTRIC FOCUSING TABLE 10 REHYDRATION SOLUTION VOLUME PER IPG STRIP IPG strip length cm Total volume per strip pl 1 125 13 250 18 390 Including sample if applied Place the IPG strip Figure 10 Remove the protective Y cover from the IPG strip ems Position the IPG strip 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 Figure 10 Overlay the IPG strip with IPG Cover Fluid Overlay each IPG strip with 1 5 to 3 ml of IPG Cover Fluid to minimize evaporation and urea crystallization 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 hours is required for rehydration overnight is recom mended If the IPG strips swell unevenly refer to Table 11 Q Prepare the Immobiline DryStrip Kit Before removing the IPG strips from the Reswelling Tray prepare the Multiphor II Immobiline DryStrip Kit and the electrode strips as described in sections 3 5 2 A and 3 5 2 B PART II FIRST DIMENSION ISOELECTRIC FOCUSING TABLE 11 TROUBLESHOOTING IPG STRIP REHYDRATION IN RESWELLING TRAY Symptom Possible cause Uneven s
8. and add SDS electrophoresis buffer See Appendix solution J 4 3 3 Electrophoresis conditions Table 22 lists the recommended conditions for the Hoefer miniVE SE 260 and SE 600 For Hoefer DALT conditions please see the User Manual Electrophoresis is performed at constant current in two steps During the initial migration and stacking period the current is approximately half of the value required for the separa tion Stop electrophoresis when the dye front is approx imately 1 mm from the bottom of the gel Cooling is optional however temperature control improves gel to gel reproducibility especially if the ambient temperature of the laboratory fluctuates signifi cantly Do not cool SDS gels below 15 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 TABLE 22 RECOMMENDED ELECTROPHORESIS CONDITIONS FOR SECOND DIMENSION VERTICAL GELS Step Current mA gel Duration h min Hoefer miniVE or SE 260 1 5 mm thick gels 1 15 0 15 TTT CN th H3 o 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 Deg re a ME m 1 0 mm thick gels 1 10 0 15 2 20 5 00 The time shown is approximate Stop electrophoresis when the dye front is 1 mm from the bottom of the gel 2Currents up to 50 higher may
9. be used if only two gels per unit are being run no divider plates and the unit is being cooled with a thermostatic circulator PART IIl 4 3 4 Troubleshooting Table 23 lists possible problems that could be encoun tered during vertical SDS PAGE and how to solve them SECOND DIMENSION SDS PAGE TABLE 23 TROUBLESHOOTING VERTICAL SECOND DIMENSION SDS PAGE Symptom Possible cause No current at Insufficient volume of buffer in upper or lower reservoir start of run Remedy Ensure that both reservoirs contain enough SDS electrophoresis buffer to contact both upper and lower electrode wires Check for leaks The second dimension SDS electrophoresis buffer is prepared incorrectly Make fresh solutions separation proceeds or resolving gel buffer is prepared incorrectly too slowly TTT Acrylamide solution is too old Prepare fresh monomer stock solution Dye front curves up Gel is not properly cooled During electrophoresis actively cool gel using a thermostatic circulator smiles at the edges che M UM a a Use the maximum possible volume of buffer in the lower reservoir Limit current to values suggested in Table 22 Dye front curves Gel is poorly polymerized near the spacers down frowns Improper instrument assembly SE 600 Leakage of upper reservoir Degas the gel solution or increase the amount of ammonium persulphate and TEMED by 50 Ensure that the gasket is not p
10. by filtration and or centrifugation Glass bead homogenization 20 21 30 The abrasive actions of the vortexed beads break cell walls liberating the cellular contents 8 USING IMMOBILIZED PH GRADIENTS Cell suspensions microorganisms Suspend cells in an equal volume of chilled lysis solution and place into a sturdy tube Add 1 3 grams of chilled glass beads per gram of wet cells Vortex 1 minute and incubate cells on ice 1 minute Repeat vortexing and chilling two to four times PART I 2 2 Protection against proteolysis When cells are lysed proteases are often liberated or acti vated Degradation of proteins through protease action greatly complicates the analysis of 2 D electrophoresis results so measures should be taken to avoid this prob lem If possible inhibit proteases by disrupting the sample directly into strong denaturants such as 8 M urea 10 TCA or 2 SDS 31 35 Proteases are less active at lower temperatures so sample preparation at as low a temperature as possible is recommended In addition most tissue proteases are inactive above pH 9 so prote olysis can often be inhibited by preparing the sample in the presence of tris base sodium carbonate or basic TABLE 6 PROTEASE INHIBITORS Protease inhibitor Effective against SAMPLE PREPARATION carrier ampholyte mixtures These approaches alone are often sufficient protection against proteolysis Some proteases however may retain some activit
11. composition IPG strip length and pH gradient requires an empirical determination for optimal results An approximate time is given in the example protocols provided in Table 16 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 pres ence of IPG Buffers which contribute 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 concentrations higher than 0 590 Results for larger quantities of protein SO pg to in excess of 1 mg and for samples loaded through sample applica tion wells can be improved by an extended focusing time and a more gradual ramping to the maximum voltage Note Complete focusing requires considerably more time with pH 6 11 L IPG strips than with the other pH gradients Note lt is generally preferable to program a protocol on the basis of volt hours rather than time At limiting current the actual maximum voltage attainable and the speed at which it is attained can vary depending on the conductivity of the sample and other components of the rehydration solution Because the optimal time for focusing can vary programming the protocol based on volt hours is preferred because it
12. concentration must not exceed 0 25 after dilution into the rehydration solution Additionally the concentration 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 2 4 Removal of contaminants that affect 2 D results Program a low initial voltage and increase voltage gradually Extend focusing time Reduce focusing time 2 D ELECTROPHORESIS 39 TROUBLESHOOTING TABLE 26 TROUBLESHOOTING 2 D RESULTS continued Symptom Possible cause Remedy Horizontal stripes across gel Impurities in agarose overlay or Prepare fresh agarose overlay and equilibration solution equilibration solution ee Prominent vertical streak at the point of sample application when loading IPG strips using sample cups Flatbed gel format Sample aggregation Dilute the sample and apply as a larger volume or precipitation RA Pn Program a low initial voltage and increase voltage gradually Insufficient equilibration Prolong equilibration time Add 30 glycerol and 6 M urea to the SDS equilibration buffer Vertical streaking Place application pieces at the end of the strips during second dimension electrophoresis to absorb excess water Second dimension buffer solutions pre
13. from 1 to 100 kDa Anal Biochem 166 368 379 1987 G rg 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 equilibra tion buffer Electrophoresis 8 122 124 1987 ORDERING Ordering information First dimension INFORMATION Multiphor II Immobiline DryStrip Kit focusing system and accessories 18 1018 06 18 1004 30 80 6371 84 18 1130 05 18 1102 77 18 1102 78 18 1004 35 18 1004 40 IPGphor Isoelectric focusing unit and accessories 80 6414 02 18 1004 40 Strip holders for use with Immobiline DryStrip Multiphor II Electrophoresis Unit Immobiline DryStrip Kit complete Immobiline DryStrip Reswelling Tray EPS 3501 XL Power Supply MultiTemp III Thermostatic Circulator 115 V MultiTemp III Thermostatic Circulator 230 V Sample cups 60 pk IEF electrode strips 100 pk IPGphor isoelectric focusing unit order strip holders separately IEF electrode strips 100 pk 7 cm 11 cm 13 cm 18 cm 6 pk 80 6416 11 80 6416 30 80 6416 49 80 6416 68 1 pk 80 6416 87 80 6417 06 80 6417 25 80 6417 44 Immobiline DryStrip gels 12 pk 7 cm 11 cm 13 cm 18 cm pH4 7L 17 6001 10 18 1016 60 17 6001 13 17 1233 01 pH 6 11 L 17 6001 34 17 6001 35 17 6001 36 17 6001 37 pH 3 10 L 17 6001 11 18 1016 61 17 6001 14 17 1234 01 pH 3 10 NL 17 6001 12 N A 17 6001 15 17 1235 01 IPG Buffer 1 ml
14. 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 degree of 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 hours is the product of the voltage and the hours elapsed at that voltage IEF performed under denaturing conditions gives the highest resolution and the cleanest results Complete denaturation and solubilization achieved with a mixture of urea and detergent ensure that each protein is present in only one configuration and minimizes aggregation and intermolecular interaction COOH cool cooe NH NH NH COOH coo C009 NuO Nu NH pH pl pH pl pH pl Net Charge 3 2 1 Isoelectric point pl Figure 7 PART II The original method for first dimension IEF depended on carrier ampholyte generated pH gradients in poly acrylamide tube gels 1 2 Carrier ampholytes are small soluble amphoteric molecules with a high buffering cap
15. resuspension can also be employed to prepare a concen trated protein sample from a dilute source e g plant tissues urine SAMPLE PREPARATION No precipitation technique is completely efficient and some proteins may not readily resuspend following precipitation Thus employing a precipitation step during sample preparation may 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 For an overview of precipitation techniques see 14 15 41 TABLE 7 PRECIPITATION PROCEDURES Precipitation method Ammonium sulphate 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 General procedure Prepare protein so final concentration of the protein solution is 51 mg ml in a buffer solution that is gt 50 mM and contains EDTA Slowly add ammonium sulphate to the desired percent saturation 41 and stir for 10 30 minutes Pellet proteins by centrifugation Limitations Many proteins remain soluble at high salt concentrations so this method is not recommended when total protein
16. sensitive than silver staining is a relatively simple method and more quantitative than silver Coomassie blue binds to proteins stoichiometrically so this staining method is preferable when relative amounts of protein are to be determined by densitometry The Hoefer Automated Gel Stainer automates multistep staining processes for increased convenience and repro ducibility Automated protocols 2 and 3 for example were developed to use the Amersham Pharmacia Biotech PlusOne Silver Staining Kit Protein to silver stain proteins in SDS gels This convenient adaptation gives reproducible results and sensitivity below 1 ng per band for most proteins Protocols 5 and 7 are recommended for Coomassie staining of SDS gels For complete details please refer to the Hoefer Automated Gel Stainer Protocol Guide 5 1 Blotting Second dimension gels can be blotted onto a nitrocellu lose or PVDF membrane for immunochemical detection of specific proteins or chemical microsequencing Note The plastic backing on ExcelGel products must be removed with a film remover prior to electrotransfer see Ordering information 36 USING IMMOBILIZED PH GRADIENTS AND ANALYSIS OF RESULTS 5 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 simple comparison of two gels is not always poss
17. solution is applied Check that the rehydration solution is evenly spread along the entire length of the IPG strip tr Poor representation of higher Proteolysis of sample Prepare sample in a manner that limits proteolysis and or use protease molecular weight proteins inhibitors See section 2 2 Protection against proteolysis m ie GE Oj mE p gu Du KAN ictu sten ed Poor transfer of protein from IPG strip to Employ a low current sample entry phase in the second dimension second dimension gel electrophoresis run Poor entry of sample protein during Use recommended volume of rehydration solution See Tables 10 and 15 rehydration Point streaking w yy Silver staining Dirty plates used to cast Properly wash glass plates Scavenge any excess or residual thiol reducing LI i gel or particulate material on the surface of agent with iodoacetamide before loading the IPG strips onto the second the gel DTT and other thiol reducing agents dimension gel exacerbate this effect Background smear toward bottom of gel Silver or Coomassie blue staining Staining of carrier ampholytes Use IPG Buffer as carrier ampholyte mixture Reduce concentration if necessary Background smear toward top of gel Silver staining Nucleic acids in sample Add DNase and RNase to hydrolyze nucleic acids Note The proteins DNase and RNase may appear on the 2 D map High background in top region of gel Protein cont
18. storage solution see Appendix solution I 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 gel caster PART III SECOND DIMENSION SDS PAGE TABLE 20 RECIPES FOR SINGLE PERCENTAGE GELS Preparation of stock solutions is described in the Appendix solutions E F G and H Final gel concentration 5 1 5 10 12 5 15 Monomer stock solution solution E 16 7 ml 25 ml 33 3 ml 417 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 persulphate solution H 500 yl 500 ul 500 yl 500 ul 500 yl TEMED 33 ul 33 ul 33 ul 33 ul 33 ul Total volume 100 ml 100 ml 100 ml 100 ml 100 ml Add after deaeration TABLE 21 RECIPES FOR GRADIENT GELS Preparation of stock solutions is described in the Appendix Solutions E F G and H Light solution final concentration 5 1 596 10 12 5 15 Monomer stock solution solution E 8 4 ml 12 5 ml 16 7 ml 21 0 ml 25 ml 4X Resolving gel buffer solution F 12 5 ml 12 5 ml 12 5 ml 12 5 ml 12 5 ml 1096 SDS solution G 500 yl
19. 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 electrophoresis It is always included in the 2 D 12 USING IMMOBILIZED PH GRADIENTS 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 9 13 51 53 A non ionic or zwitterionic detergent is always included in the sample solution to ensure complete sample solu bilization and to prevent aggregation through hydro phobic interactions Originally either of two similar non ionic detergents NP 40 or Triton X 100 were used 1 2 Subsequent studies have demonstrated that the zwitterionic detergent CHAPS is often more effective 54 Non ionic or zwitterionic detergents are used in concentrations up to 4 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 PART l SAMPLE with proteins it cannot be used as the sole detergent for solubilizing samples for 2 D electrophoresis A widely used method for
20. 0 01 1 2 3500 2 5 1 30 2900 3 3500 2 5 3 45 4 20 13100 18100 Total 5 15 5 50 16000 21000 13 cm pH 6 11 L pH 3 10 L and pH 3 10 NL 1 300 2 5 0 01 1 2 3500 2 5 1 30 2900 3 3500 2 5 3 10 4 00 11100 14100 Total 4 40 5 30 14000 17000 18 cm pH 4 71 1 500 2 5 0 01 1 2 3500 2 5 1 30 3000 3 3500 2 5 5 40 7 40 20000 27000 Total 7 10 9 10 23000 30000 18 cm pH 6 11 L pH 3 10 L and pH 3 10 NL 1 500 2 5 0 01 1 2 3500 2 5 1 30 3000 3 3500 2 5 4 50 6 20 17000 22000 Total 6 20 7 50 20000 25000 During phase 2 the voltage will rise from the voltage set for phase 1 to 3500 V The voltage will remain at 3500 V throughout phase 3 2When applying sample onto pH 6 11 L IPG strips by inclusion in the rehydration solution more time is required for complete focusing Increase the recommended volt hours Vh in the final phase of the program by 6 to 10 fold for 7 cm long IPG strips 5 to 8 fold for 11 cm long IPG strips and 5 to 7 fold for 13 and 18 cm long IPG strips 22 USING IMMOBILIZED PH GRADIENTS PART II 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 After IEF proceed to the second dimension separation immediately or store the IPG strips at 40 to 80 C in FIRST DIMENSION ISOELECTRIC FOCUSING screw cap
21. 1 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 MultiTemp III Thermostatic Circulator 4 USING IMMOBILIZED PH GRADIENTS The IPGphor Isoelectric Focusing System Figure 2 further simplifies the first dimension separation with a system dedicated to IEF separation on IPG strips The system is comprised of IPG strip holders that serve as both 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 IPGphor platform for any one protocol Because rehydration and IEF are performed consecutively with out user intervention they can be performed unattended overnight 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 Table 2 shows the key operating differences between the Multiphor II system and the IPGphor Isoelectric Focusing System for first dimension IEF TABLE 2 IEF SYSTEM SELECTION Maximum Additional Time required voltage eq
22. 17 6000 86 17 6000 87 17 6000 88 17 6001 78 pH 4 7 L pH 3 10 L pH 3 10 NL pH 6 11 L Pharmalyte 25 ml 17 0453 01 17 0455 01 pH 5 8 pH 8 10 5 2 D ELECTROPHORESIS 47 ORDERING INFORMATION Second dimension 18 1124 82 2 D Electrophoresis brochure Hoefer mini vertical units and accessories 80 6418 77 80 6150 11 80 6150 30 80 6149 35 80 6146 12 80 6146 50 80 6223 83 80 6127 88 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 10 x 10 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 unit and accessories 80 6171 58 80 6179 94 80 6180 70 80 6180 13 80 6180 89 80 6179 18 80 6182 79 SE 600 Dual Cooled Vertical Slab Unit for up to 4 gels glass plate size 18 x 16 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 18 x 16 cm notched SE 615 Multiple Gel Caster for 2 to 10 gels glass plate size 18 x 16 cm Hoefer DALT vertical unit and accessories 80 6068 79 80 6068 98 80 6330 61 80 6067 27 80 6067 46 80 6067 65 80 6067 84 Gradient makers 80
23. 2 D Electrophoresis USING IMMOBILIZED PH GRADIENTS PRINCIPLES amp METHODS 80 6429 60 e Rev A 9 98 amersham pharmacia biotech Easy Breeze ExcelGel Hoefer Immobiline IPGphor Multiphor MultiTemp Pharmalyte PlusOne and Ultrodex are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia and Upjohn Inc All other trademarks and registered trademarks are the property of their respective companies or organizations Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech AB SE 751 84 Uppsala Sweden Amersham Pharmacia Biotech Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Pharmacia Biotech Inc 1998 All rights reserved All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them A copy of these terms and conditions is available on request 2 D Electrophoresis USING IMMOBILIZED PH GRADIENTS PRINCIPLES amp METHODS Tom Berkelman and Tirra Stenstedt with contributions from Bengt Bjellqvist Nancy Laird Ingmar Olsson Wayne Stochaj Reiner Westermeier Preface Proteomics is the large scale screening of the proteins of a cell organ ism or biological fluid a process which requires stringe
24. 318 01 17 1329 01 80 1130 01 17 1335 01 17 1302 01 17 1302 02 17 1300 01 17 1300 02 17 1303 01 17 1301 01 17 1304 01 17 1306 01 17 1321 01 17 1313 01 17 1325 01 17 1311 01 17 1312 01 17 1323 01 17 0422 01 17 0554 01 Enzymes 27 0516 01 27 0330 02 27 0323 01 Urea 500 g CHAPS 1 g Triton X 100 500 ml Dithiothreitol DTT 1 g Bromophenol blue 10 g Ultrodex granulated gel 50 g IPG Cover Fluid 1 000 ml Acrylamide PAGE acrylic acid lt 0 05 250 g as above 1 kg Acrylamide IEF acrylic acid lt 0 002 250 g as above 1 kg Acrylamide PAGE 40 solution 1 000 ml Acrylamide IEF 40 solution 1 000 ml N N methylenebisacrylamide 25 g N N methylenebisacrylamide 2 solution 1 000 ml Tris 500 g SDS 100 g Glycerol 87 1 L Ammonium persulphate 25 g TEMED 25 ml Glycine 500 g Agarose M 10 g Agarose NA 10 g Deoxyribonuclease I DNase I 20 mg Ribonuclease I RNase A and RNase B 1 g Ribonuclease I A RNase A 100 mg Molecular weight markers 80 1129 83 17 0446 01 80 1129 46 MW range 2 512 16 949 MW range 14 400 94 000 IEF sample application pieces 200 pk 2 D ELECTROPHORESIS 49 ORDERING INFORMATION pl calibration kits 17 0582 01 Carbamylyte Calibration Kit Automated gel staining 17 1150 01 80 6395 02 80 6396 16 17 0518 01 80 6343 34 Gel driers 80 6121 61 80 6121 80 80 6428 84 80 6429 03 Silver Staining Kit Protein Hoefer Auto
25. 500 yl 500 yl 500 ul 500 yl Double distilled water 28 5 ml 24 5 ml 20 1 ml 16 0 ml 12 0 ml 10 Ammonium persulphate solution H 165 yl 165 yl 165 yl 165 yl 165 yl TEMED 16 5 ul 16 5 yl 16 5 ul 16 5 yl 16 5 yl Total volume 50 ml 50 ml 50 ml 50 ml 50 ml Heavy solution final concentration 10 12 5 15 17 5 20 Monomer stock solution solution E 16 7 ml 21 0 ml 25 0 ml 29 2 ml 33 3 ml 4X Resolving gel buffer solution F 12 5 ml 12 5 ml 12 5 ml 12 5 ml 12 5 ml Sucrose 15g 15g 15g 15g 15g 1076 SDS solution G 500 yl 500 yl 500 yl 500 ul 500 yl Double distilled water 16 2 ml 11 7 ml 7 1 ml 3 5 ml 0 ml 10 Ammonium persulphate solution H 165 yl 165 yl 165 yl 165 yl 165 yl TEMED 16 5 yl 16 5 yl 16 5 yl 16 5 yl 16 5 ul Total volume 50 ml 50 ml 50 ml 50 ml 50 ml Add after deaeration 2 D ELECTROPHORESIS 31 PART IIl SECOND DIMENSION SDS PAGE Figure 19 Figure 20 4 3 2 Applying the equilibrated IPG strip See section 4 2 2 for equilibration protocol Place the IPG strip Dip the IPG strip from section 4 2 2 in the SDS elec trophoresis buffer see Appendix solution J to lubricate it 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 Figure 19 With a thin plastic ruler gently push the IPG strip down so the entire lower edge of the IPG strip is in contact with the top surface of the slab gel Figure 20 Ensure t
26. 6197 80 80 6197 99 80 6196 09 80 6198 18 Multiphor II 18 1018 06 80 1129 46 18 1013 75 Power supplies 80 6406 99 18 1130 01 18 1130 02 18 1130 03 DALT Multiple Cooled Vertical Slab Gel Unit with buffer circulation pump for up to 10 gels gel cassette size 25 x 20 cm 115 V Same as above 230 V DALT Multiple Gel Caster for 23 gels DALT Cassette with 1 0 mm spacers DALT Cassette with 1 5 mm spacers DALT Gradient Maker with peristaltic pump 115 V DALT Gradient Maker with peristaltic pump 230 V SG 30 Gradient Maker 30 ml total volume SG 50 Gradient Maker 50 ml total volume SG 100 Gradient Maker 100 ml total volume SG 500 Gradient Maker 500 ml total volume Multiphor II Electrophoresis Unit IEF sample application pieces 200 pk Film remover for electrophoretic transfer EPS 2A200 Power Supply 200 V 2 000 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 1 000 V 400 mA 100 W 48 USING IMMOBILIZED PH GRADIENTS ORDERING INFORMATION Thermostatic circulator 18 1102 77 18 1102 78 MultiTemp III Thermostatic Circulator 115 V MultiTemp III Thermostatic Circulator 230 V ExcelGel SDS gradient gels 80 1255 53 17 1236 01 17 1342 01 ExcelGel SDS 8 18 6 pk ExcelGel SDS XL 12 14 3 pk ExcelGel SDS Buffer Strips anode and cathode 6 each pk PlusOne electrophoresis chemicals and reagents 17 1319 01 17 1314 01 17 1315 01 17 1
27. A EN OE aa FN TA AKA Sen NEG E TO is E memo AGRON OM MION aaa 9 Prepare the sample Prepare the sample in a solution similar in composition to the rehydration solution used E Determine the point of sample application The optimal application point depends on the charac teristics 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 gradients and preferred when pH 3 10 gradients are used The optimal application point can vary with the nature of the sample Empirical determi nation of the optimal application point is best Position the sample cup bar Place sample cups on the sample cup bar high enough on the bar to avoid touching the gel surface Position the sample cup bar so that the sample cups are a few millime ters away from the cathodic or anodic electrode depend ing on your sample The sample cups must face the electrode The sample cup bar has a spacer on one side Slide the sample cup bar toward the anode cathode until the spacer just touches the anodic cathodic electrode PART II Press the sample cups against the IPG strips Fig 13 ty Move the sample cups into position one sample cup ac above each IPG strip and TW ext _ Press the sample cups down he to ensure good contact with 3 each IPG strip This is i perhaps the most critic
28. CTROPHORESIS 27 PART IIl Part II Second dimension SDS PAGE 4 0 Second dimension SDS PAGE overview After IEF the second dimension separation can be performed on various flatbed or vertical systems depend ing on factors such as those discussed in section 1 2 Equipment choices 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 4 3 and 4 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 4 1 Background to SDS PAGE SDS PAGE SDS polyacrylamide gel electrophoresis is an electrophoretic method for separating polypeptides according to their molecular weights MW The tech nique is performed in polyacrylamide gels containing sodium dodecyl sulphate SDS The intrinsic electrical charge of the sample proteins is not a factor in the sepa ration due to the presence of SDS in the sample and the gel SDS is an anionic detergent that denatures proteins
29. Focusing System 3 1 Background to isoelectric focusing IEF IEF is an electrophoretic method that separates proteins according to their isoelectric points pl Proteins are amphoteric molecules they carry either positive negative or zero net charge depending on the pH of their surroundings see Figure 7 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 is the specific pH at which the net charge of the protein is zero Proteins are positively charged at pH values below their pI and negatively charged at pH values above their pl If the net charge of a protein is plotted versus the pH of its environment see Figure 7 the resulting curve intersects the abscissa at the isoelectric point 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 14 USING IMMOBILIZED PH GRADIENTS FIRST DIMENSION ISOELECTRIC FOCUSING 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 pl 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
30. PG strips so that they rest on an edge IPG strips can be left in this position for up to 10 minutes without noticeably affecting the spot sharpness Alternatively the IPG strips can be gently blotted with moistened filter paper to remove excess equilibration buffer 4 3 Vertical systems 4 3 1 Preparing SDS slab gels vertical systems The instructions provided below for the preparation of vertical SDS polyacrylamide gels employ the tris glycine system of Laemmli 63 Vertical second dimension gels are most conveniently cast several at a time in a multi ple gel caster see Ordering information For assembly of the gel cassette refer to the relevant User Manual O Select the gel percentage a Single percentage gel versus gradient gel Single percentage gels offer better resolution for a particular MW window A commonly used second dimension gel for 2 D electrophoresis is a homoge neous gel containing 12 5 total acrylamide When a gradient gel is used the overall separation interval is wider and the linear separation interval is larger In addition bands are sharper because the decreasing pore size functions to minimize diffusion A gradient gel requires more skill to cast however For detailed instructions on gradient preparation see the User Manual for the relevant gel unit gradient maker and gel caster Note Stacking gels are not necessary for vertical 2 D gels 2 D ELECTROPHORESIS 29 PART IIl b Whe
31. PLE PREPARATION TABLE 8 CONTAMINANTS THAT AFFECT 2 D RESULTS continued Contaminant Reason for removal Removal techniques Polysaccharides Polysaccharides can clog gel pores causing either precipitation or Precipitate the sample in TCA ammonium sulphate or extended focusing times and resulting in horizontal streaking phenol ammonium acetate then centrifuge Some polysaccharides contain negative charges and can complex Ultracentrifugation will remove high molecular weight with proteins by electrostatic interactions polysaccharides Employing the same methods used for preventing protein nucleic acid interactions may also be helpful solubilize sample in SDS or at high pH Lipids Many proteins particularly membrane proteins are complexed with Strongly denaturing conditions and detergents minimize 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 protein lipid interactions Excess detergent may be necessary Precipitation with acetone removes some lipid lipid layer that can be difficult to remove Phenolic compounds 40 46 Phenolic compounds are present in many plant tissues and can modify proteins through an enzyme catalyzed oxidative reaction Prevent phenolic oxidation by employing
32. S electrophoresis buffer 25 mM Tris 192 mM glycine 0 1 SDS 5 liters Final concentration Amount Wis base FW 1211 25mM NUTI Glycine FW 75 07 192 mM 121g SDS FW 288 38 o DI CO OB Double distilled H 0 to 5000 ml Store at room temperature Because the pH of this solution need not be checked it can be made up directly in large reagent bottles marked at 5 0 liters 20 liters can be made up at a time and stored at room temperature K Agarose sealing solution Final concentration SDS electrophoresis buffer see solution Agarose NA or M 0 5 Bromophenol blue trace Amount ME 0 5 g a few grains Add all ingredients into a 500 ml Erlenmeyer flask Swirl to disperse Heat in a microwave oven on low 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 2 D ELECTROPHORESIS 43 REFERENCES References O Farrell P H High resolution two dimensional electrophoresis 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 G rg A Postel W G nther S Weser J Improved horizontal two dimensional electrophoresis with hybrid isoelectric focusing in immobilized pH gradi ents in the first dimension and l
33. 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 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 pl nor leave the sample in a highly conductive solution In general concentrated urea as well as one or more detergents are used Sample solution composition is discussed in section 2 5 PART I General sample preparation guidelines Keep the sample preparation strategy as simple as possi ble to avoid protein losses Additional sample prepara tion steps may improve the quality of the final 2 D result but at the possible expense of selective protein loss 1 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 Sample preparation solutions should be freshly prepared or stored as frozen aliquots Use high purity or de ionized urea Preserve sample quality by preparing the sample just prior to IEF or storing samples in aliquots at 80 C
34. a can introduce protein charge modifications Sonication helps speed solubilization particularly from material that is otherwise difficult to resuspend A widely used sample solution is the lysis solution given in the Appendix solution A For a general review of protein solubilization for elec trophoretic analysis see 12 PREPARATION 2 D ELECTROPHORESIS 13 PART II Part lI First dimension isoelectric focusing 3 0 First dimension isoelectric focusing overview Amersham Pharmacia Biotech offers two different systems for the first dimension separation the Multi phor II system with associated accessories and the IPGphor Isoelectric Focusing System A comparison of these two systems is given in section 1 2 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 3 2 Sample application methods and their selection are discussed in section 3 3 The first dimension separation procedure involves IPG strip rehydration sample application and isoelectric focusing Preparation of the IPG strip rehydration solu tion is described in section 3 4 The protocols for IPG strip rehydration sample application and IEF are specific to the first dimension system used and are described in section 3 5 for the Multiphor II system and section 3 6 for the IPGphor Isoelectric
35. acity near their pl Commercial carrier ampholyte mixtures are comprised of hundreds of individual poly meric species with pls spanning a specific pH range When a voltage is applied across a carrier ampholyte mixture the carrier ampholytes with the lowest pI and the most negative charge move toward the anode and the carrier ampholytes with the highest 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 envi ronment to the corresponding pHs The result is a continuous pH gradient Although this basic method has been used in hundreds of 2 D electrophoresis studies it has several limitations that have prevented its more widespread application 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 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 The soft polyacrylamide tube gels have low mechanical stability The gel rods may stretch or break affecting reproducibility Results ar
36. age A notch at the lower left corner of the film identifies the 18 or 14 i e anodic end Note The gel is cast on a plastic support film and does not cover the film entirely Markings on the plastic cover of the gel indicate the direction of electrophoresis Orient the gel according to these markings remove the cover and place the gel on the cooling plate Note Avoid trapping bubbles between the gel and the cooling plate Avoid getting IPG Cover Fluid or kerosene on the gel surface as this may cause the buffer strips to slide during electrophoresis Separation quality is improved if the gel surface is allowed to dry uncovered for about 5 minutes before proceeding Position the cathodic buffer strip Figure 22 Peel back the foil on the colorless cathodic ExcelGel SDS buffer strip Place the buffer strip with the smooth narrow face downward along and in complete contact 2 D ELECTROPHORESIS 33 PART IIl with the cathodic edge of the SDS gel Avoid trapping air bubbles between the gel and the buffer strip 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 Q Position the anodic buffer strip Repeat step 4 with the yellow colored anodic Excel Gel buffer strip placing it along and in contact with the anodic edg
37. al part of the setup Check that the strips are in their correct straight position in the DryStrip aligner 3 d at E TAU f ma Figure 13 Apply IPG Cover Fluid Once the sample cups are properly positioned pour 70 to 80 ml of IPG Cover Fluid into the tray to completely cover the IPG strips If the IPG Cover Fluid leaks into the sample cups correct the position of the sample cups remove the fluid from the cups with a pipette and check for leakage again Add approximately 150 ml of addi tional IPG Cover Fluid to cover the sample cups The IPG strips are submerged under a layer of IPG Cover Fluid to prevent drying of the IPG strip precipitation of the components of the rehydration solution and diffu sion of gasses into the IPG strip Q Apply the sample Figure 14 MAT a Apply sample up to EC Fu 100 pl per IPG strip ai into the sample cups by pipetting under the surface of the IPG Cover Fluid The sample should sink to the bottom of the cup Watch for leakage Figure 14 Note As mentioned in section 3 3 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 had been included in the rehydration solution These limitations can sometimes be minimized with the following suggestions Protein precipitation and aggregation at the application point can sometimes be avoided 1 The sample should contain
38. aminant in SDS electrophoresis buffer or dirty electrophoresis unit Make fresh SDS electrophoresis buffer Clean electrophoresis unit 2 D ELECTROPHORESIS 41 APPENDIX Appendix Solutions A Lysis solution 8 M urea 4 CHAPS 40 mM Tris base 40 ml Final concentration Amount Urea PW 60 06 ML c M CHAPS MA ERE Leu TisbaeW12L OM OM Double distilled H20 to 40 ml Prepare fresh or store in aliquots at 20 C f 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 Note Protease inhibitors and or reductants may be added if necessary B Rehydration stock solution without IPG Buffer 8 M urea 2 CHAPS bromophenol blue 25 ml Final concentration Amount Urea FPW 60 06 OM Lee IE TE Bromophenolbue trace 2 few grains Double distilled H 0 to 25 ml Store in 2 5 ml aliquots at 20 C DIT and IPG Buffer are added just prior to use Add 7 mg DTT per 2 5 ml aliquot of rehydration stock solution See Table 9 for the appropriate volume of IPG Buffer to use If loading sample by inclusion in the rehydration solution sample is also added to the 2 5 ml aliquot of rehydration solution just prior to use If necessary the concentration of urea can be increased to 9 or 9 8 M 3 Other detergents Triton X 100 NP 40 and other non ionic or zwitterio
39. applied to the reservoir slots of the Reswelling Tray or the IPGphor strip holders then the IPG strips are soaked individually Rehydrated strips are 3 mm wide and approximately 0 5 mm thick 3 4 1 Components of the rehydration solution Selection of the optimal rehydration solution will depend on the specific protein solubility requirements of the sample A typical solution generally contains urea non ionic or zwitterionic detergent dithiothreitol DTT IPG Buffer Amersham Pharmacia Biotech 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 recom mended concentration range gt 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 It has recently been reported that using thiourea in addition to urea further improves solubilization particularly of membrane proteins 9 13 51 53 gt Detergent solubilizes hydrophobic proteins and mini mizes protein aggregation The detergent must have zero net charge use only non ionic and zwitterionic detergents CHAPS Triton X 100 or NP 40 in a concentration of 0 5 to 4 are most commonly used gt Reductant cleaves disulphide bonds to allow proteins to unfold completely DTT or DTE 20 to 100 mM is commonly used 2 Mercap
40. ation solution Usually IPG IEF starts close to 1 mA and drops into the pA range This depends on the number of IPG strips in the instrument Make sure that the low current shut off has been bypassed see power supply instructions IPG IEF may start in a current range that is not detectable by the power supply Always include IPG Buffer in the rehydration solution No current at start of run No electrode contact or lack of electrical continuity IPG strip is improperly rehydrated The high voltage ead from the electrophoresis unit is not plugged into the power supply correctly Check to make sure that all Multiphor ll contacts are in place Make sure that 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 Sample dye does not move out of the sample cup 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 Thi
41. ause the precast Immobiline DryStrip IPG strips are highly reproducible the pI of a particular protein can be estimated from its focusing position along a linear pH gradient IPG strip The second dimension can be calibrated using molecular weight marker proteins loaded to the side of the second dimension gel Often there are abundant proteins in the sample for which the pI and molecular weight are known These proteins can serve as internal standards Note The pl of a protein can depend on its chemical environment and thus can differ depending on the exper imental conditions used Although marker proteins for pl estimation are available pI estimates based on their use are therefore not necessarily valid TROUBLESHOOTING Troubleshooting 6 0 Troubleshooting 2 D results Table 26 lists problems that may be encountered in 2 D electrophoresis results describes the possible causes and suggests ways to prevent each problem in future experiments For troubleshooting problems encountered during the various steps of the 2 D process refer to the following tables Table 11 page 19 Troubleshooting IPG strip rehy dration in Reswelling Tray TABLE 26 TROUBLESHOOTING 2 D RESULTS Table 14 page 23 Troubleshooting first dimension IEF Multiphor II and Immobiline DryStrip Kit Table 17 page 27 Troubleshooting first dimension IEF IPGphor 1 Table 23 page 33 Troubleshooting vertical second dimension SDS PAGE 1 Table 25 pa
42. aying on transfer to the second dimension Electrophoresis 6 599 604 1985 G rg 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 Bio Technol ogy 14 61 65 1996 Pennington S R Wilkins M R Hochstrasser D E Dunn M J Proteome analysis From protein charac terization to biological function Trends in Cell Biol ogy 7 168 173 1997 Gorg A Boguth G Obermaier C Posch A Weiss W Two dimensional polyacrylamide gel elec trophoresis 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 L Traini M Sanchez J C Hochstrasser D F Williams K L Gooley A A Extraction of mem brane proteins by differential solubilization for sepa ration using two dimensional gel e
43. bstances Table 8 lists contaminants that affect 2 D results and techniques for their removal Reference 12 provides further discussion on the removal of interfering substances TABLE 8 CONTAMINANTS THAT AFFECT 2 D RESULTS Contaminant Reason for removal Salts residual buffers and other Salts disturb the electrophoresis process and must be removed or charged small molecules that carry maintained at as low a concentration as possible over from sample preparation 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 in the final result 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 to 50 mM in the sample may be tolerated however proteins may precipitate at the sample application point as they abruptly move into a lower salt environment 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 resu
44. by gradually increasing the voltage across the IPG strips to at least 3 500 V and maintaining this volt age 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 separa tion the resultant 2 D maps are superior in terms of resolution and reproducibility IPG strips are a marked improvement over the tube gels with carrier ampholyte generated pH gradients 1 The first dimension separation is more reproducible because the covalently fixed gradient cannot drift 1 The plastic backed IPG strips are easy to handle They can be picked up at either end with forceps or gloved fingers 1 The plastic support film prevents the gels from stretch ing or breaking 1 PG technology increases the useful pH range on any single IPG strip more very acidic and basic proteins can be separated The IPG strips have a higher loading capacity for protein 59 1 The sample can be introduced into the IPG strip during rehydration 60 61 Precast Immobiline DryStrip gels are available from Amersham Pharmacia Biotech These ready made dry IPG strips eliminate the need to handle toxic acry lamide monomers preparation time and effort are significantly reduced and reproducibility of the pH gradient is assured 3 2 Immobilized pH gradient selection Ready made IPG strips Immobiline DrySt
45. by wrapping around the polypeptide backbone in a ratio of approximately 1 4 grams SDS per gram protein The bound SDS masks the charge of the proteins themselves forming anionic complexes with constant net negative charge per unit mass The SDS also disrupts hydrogen bonds blocks hydrophobic interactions and partially unfolds the protein molecules minimizing differences in molecular form by eliminating the tertiary and secondary structures The proteins are totally unfolded when a reducing agent such as DTT is employed The disulphide bonds which can form between cysteine residues are cleaved and the polypeptides become flexible rods of negative charges with equal charge densities or charge per unit length When proteins are treated with both SDS and a reducing agent separations exclusively by molecu 28 USING IMMOBILIZED PH GRADIENTS SECOND DIMENSION SDS PAGE lar weight are possible In fact there is an approximately linear relationship between the logarithm of the molecu lar weight and the relative distance of migration of the SDS polypeptide micelle Note This linear relationship is valid only for a certain molecular weight range that 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 63 Other buffer systems can be used particularly the tris tricine system of Schigger and von Jagow 64 for resolution
46. cess 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 Prepare for electrophoresis Remove the rehydrated IPG strip from the Reswelling Tray To remove an IPG strip from its slot in 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 2 D ELECTROPHORESIS 19 PART II Rinse the IPG strip with deionized water Hold the IPG strip with the forceps and rinse briefly in a stream of deionized water delivered from a squeeze bottle This rinse will remove excess rehydration solution and thus prevent formation of urea crystals on the gel surface during IEE Place the IPG strip on its edge on a damp filter paper for several seconds to drain excess moisture Avoid contact between the gel surface and the filter paper Position the IPG strip in the DryStrip aligner Fig 11 Immediately trans fer the rehydrated IPG strips to adja cent 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 that the anodic gel edges are lined up Figure 11
47. chelating free Peptide protease inhibitors These inhibitors are e reversible inhibitors e active in the presence of DIT e active at low concentrations under a variety of conditions Use at 2 20 ug ml as pepsin Aprotinin inhibits many serine proteases Bestatin inhibits aminopeptidases Leupeptin inhibits many serine and cysteine proteases e g leupeptin pepstatin aprotinin bestatin pepstatin inhibits aspartyl proteases e g acidic proteases such Peptide protease inhibitors are expensive Peptide protease inhibitors are small peptides and thus may appear on the 2 D map depending on the size range separated by the second dimension gel Pepstatin does not inhibit any proteases that are active at pH 9 TLCK TPCK Tosyl lysine chloromethyl ketone tosyl phenylalanine chloromethyl ketone Use at 0 1 0 5 mM cysteine proteases These similar compounds irreversibly inhibit many serine and Benzamidine Use at 1 3 mM Benzamidine inhibits serine proteases 2 D ELECTROPHORESIS 9 PART I 2 3 Precipitation procedures Protein precipitation is an optional step in sample preparation for 2 D electrophoresis Precipitation followed by resuspension in sample solution is gener ally employed to selectively separate proteins in the sample from contaminating species such as salts deter gents nucleic acids lipids etc that would otherwise interfere with the 2 D result Precipitation followed by
48. cia products Tel 0169 35 67 00 Fax 0169 41 96 77 Germany Tel 07 61 49 03 0 Fax 07 61 49 03 405 Italy Tel 02 27322 1 Fax 02 27302 212 Japan Tel 81 3 5331 9317 Fax 81 3 5331 9372 Latin America Tel 55 11 3667 5700 Fax 55 11 3667 5899 Middle East and Africa Tel 30 1 96 00 687 Fax 30 1 96 00 693 Netherlands Tel 0165 580 410 Fax 0165 580 401 Norway Tel 47 63 89 23 10 Fax 47 63 89 23 15 Portugal Tel 01 417 70 35 Fax 01 417 31 84 South East Asia Tel 60 3 724 2080 Fax 60 3 724 2090 South East Europe Tel 43 1 982 3826 Fax 43 1 985 8327 Spain Tel 935 944 950 Fax 935 944 955 Sweden Tel 018 16 40 00 Fax 018 71 24 44 Switzerland Tel 01 802 81 50 Fax 01 802 81 51 UK Tel 0800 515 313 Fax 0800 616 927 USA Tel 1 800 526 3593 Fax 1 800 329 3593
49. cially convenient if temperature control during rehydration is a concern 3 6 2 Optional Apply electrode pads Under certain conditions such as prolonged focusing water may migrate toward one end of the IPG strip causing the other end to begin drying out This effect can PART II be minimized by placing paper electrode pads between the IPG strip and each strip holder electrode just before IEF Electrode pads may also absorb ions that would otherwise accumulate at the ends of the IPG strip and possibly interfere with the separation O Prepare electrode pads 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 filter paper Important Electrode pads must be damp not wet Position electrode pads Remove cover from strip holder Using forceps or tweez ers 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 Replace cover on strip holder 3 6 3 Optional Apply sample after gel rehydration If the sample was not applied as a part of the rehydra tion solution it can be applied immediately prior to IEF O Prepare sample Prepare the sample in a solution similar in composition to the rehydration solution used O Apply sample Figure 18 p Remove cover from strip holder Pipet
50. compensates for this variability Note Exceeding the current limit of 50 pA per 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 is seldom a problem below 100 000 total volt hours but on longer runs it may contribute to 2 D ELECTROPHORESIS 25 PART II FIRST DIMENSION ISOELECTRIC FOCUSING TABLE 16 IMMOBILINE DRYSTRIP IEF GUIDELINES horizontal streaking visible in the 2 D result See also FOR IPGPHOR ISOELECTRIC FOCUSING SYSTEM section 6 0 Troubleshooting 2 D results S0 p per IPA strip 3 6 5 Protocol examples IPGphor 20 C for both rehydration and IEF The protocols given in Table 16 are suitable for first pH gradients 4 7 L 3 10 L and 3 10 NL dimension isoelectric focusing of protein samples BUND OUTRE Waltnours EEG suspended in rehydration solution in typical analytical Step Voltage h min Vh type quantities 1 to 50 ng The protocols are optimized for a rehydration solution containing 0 5 IPG Buffer The LEI skalio sx FONOJ recommended current limit is 50 nA per IPG strip 3 1000 030 500 a Recommended focusing times are given but the optimal 4 8000 1 00 8000 Step n hold length of time will depend on the nature of the sample the amount of protein and the method of sample appli We ues pen a mm cation Please refer to the IPGpho
51. cts from plants Methods Enzymol 182 174 193 1990 Theillet C Delpeyroux E 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 Blomberg 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 elec trophoresis 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 varia tion detected in wheat seedling proteins Elec trophoresis 7 52 54 1986 Wu ES Wang M Y Extraction of proteins for sodium dodecyl sulfate polyacrylamide gel elec trophoresis from protease rich plant tissues Amal Biochem 139 100 103 1984 Harrison P A Black C C Two dimensional elec trophoretic 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 nebu
52. d analysis can be performed on increasingly smaller samples Immunochemical identification is now possible with a wide assortment of available antibodies More powerful less expensive computers and software are now available allowing routine computerized eval uations of the highly complex 2 D patterns 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 The World Wide Web provides simple direct access to spot pattern databases for the comparison of elec trophoresis results and to genome sequence databases for assignment of sequence information A large and growing application of 2 D electrophoresis is proteome analysis Proteome analysis is the analysis of the PROTEin complement expressed by a genOME 5 6 The analysis involves the systematic separation identification and quantification of many proteins simul taneously from a single sample 2 D electrophoresis is used in this application due to its unparalleled ability to separate thousands of proteins 2 D electrophoresis is also unique in its ability to detect post and cotransla tional modifications which cannot be predicted from the genome sequence Other applications of 2 D electrophoresis include analy sis of cell differentiation detection of disease markers monitoring therapies drug discovery cancer re
53. d section 2 5 for details Enzymatic lysis 20 21 Cells with cell walls can be lysed gently following enzymatic removal of the cell wall This must be done with an enzyme specific for the type of cell to be lysed e g lysozyme for bacterial cells cellulase and pectinase for plant cells lyticase for yeast cells Plant tissue bacterial cells fungal cells Treat cells with enzyme in isoosmotic solution 2 D ELECTROPHORESIS 7 PART I lysed cells such as tissue culture cells blood cells and some microorganisms Gentle lysis methods can also be employed when only one particular subcellular fraction is to be analyzed For example conditions can be chosen in which only cytoplasmic proteins are released or intact mitochondria or other organelles are recovered by differ ential centrifugation Sometimes these techniques are combined e g osmotic lysis following enzymatic treat ment freeze thaw in the presence of detergent TABLE 5 MORE VIGOROUS LYSIS METHODS Cell disruption method Sonication 4 22 23 Ultrasonic waves generated by a sonicator lyse cells through shear forces Complete shearing is obtained when maximal agitation is achieved but care must be taken to minimize heating and foaming SAMPLE Application Cell suspensions PREPARATION 2 1 2 More vigorous lysis methods More vigorous lysis methods listed in Table 5 are employed when cells are less easily disrupted i e cells in solid tis
54. e 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 IPGphor protocol settings are incorrect for Check that the current limit is properly set th iment ies mi eh PEPENE Check that the actual number of strips on the IPGphor platform equals the number of maximum set value s strips entered in the protocol Conductivitfonicstrength is too high Prepare the sample to yield a salt concentration less than 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 setting of 50 pA per IPG strip inthe strips 0 gogna Ensure that 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 that the entire IPG strip surface is wetted The IPG strip dried during IEF Always apply IPG Cover Fluid to prevent dehydration of a rehydrated IPG strip 2 D ELE
55. e of the SDS gel 4 4 2 Applying the equilibrated IPG strip See section 4 2 2 for the equilibration protocol Place the IPG strip s Figure 23 Once the equilibrated IPG strips from section 4 2 2 have drained for at least 3 minutes place the IPG strips gel side down on the SDS gel so that the cathodic buffer strip and the IPG strip are parallel to each other and 2 to 3 mm apart Place sample application pieces Figure 24 Place one IEF sample application piece on the SDS gel underneath the plastic tab formed by the overhanging gel support film at each end of the IPG strip s Be sure the application pieces are positioned so that they touch the ends of the IPG strip Note Application pieces absorb water that flows out of the IPG strips during electrophoresis 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 pair of forceps 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 SECOND DIMENSION SDS PAGE IPG strip Pipette the markers onto the extra sample ap plication piece Apply the markers in a volume of 15 to 20 pl For less volume cut the sample application piece proportionally The markers should contain 200 to 1 000 ng of each component f
56. e often dependent on the skill of the operator Because of the limitations of the carrier ampholytes method an alternative technique for pH gradient forma tion was developed immobilized pH gradients or IPG This technique was introduced by Bjellqvist and others in 1982 58 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 The buffers called acrylamido buffers Amersham Pharmacia Biotech Immobiline reagents are a set of well characterized molecules each with a single acidic or basic buffering group linked to an acrylamide monomer Their general structure is the following CH CH C NH R ll 0 R weakly acidic or basic buffering group FIRST DIMENSION ISOELECTRIC FOCUSING Immobilized pH gradients are formed using two solu tions one containing a relatively acidic mixture of acry lamido 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 8 is a graphical representa tion of the polyacrylamide matrix with attached buffer ing groups Figure 8 For improved perfo
57. eficiency in maize VI Changes in the two dimensional elec trophoretic 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 Resolu tion of fibronectin and other uncharacterized proteins by two dimensional polyacryamide elec trophoresis with thiourea J Cbromat 705 351 356 1997 Pasquali C Fialka I Huber L A Preparative two dimensional gel electrophoresis of membrane proteins Electrophoresis 18 2573 2581 1997 Rabilloud T Use of thiourea to increase the solubil ity of membrane proteins in two dimensional elec trophoresis 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 Wilson D L Hall M E Stone G C Rubin R W Some improvements in two dimensional gel elec trophoresis 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 Electrophore sis 19 845 851 1998 Bjellqvis
58. election 16 3 4 IPG strip rehydration solution 17 3 4 1 Components of the rehydration solution 17 3 4 2 Rehydration solution preparation 18 3 5 Multiphor II and Immobiline DryStrip Kit 18 3 5 1 IPG strip rehydration Immobiline DryStrip Reswelling Tray 18 3 5 2 Preparing for IEF iiinc 19 A Prepare the Immobiline DryStrip Kit 19 B Prepare electrode strips 19 C Prepare for electrophoresis 19 D Optional Apply sample after gel rehydration sssssssss 20 3 5 3 Isoelectric focusing guidelines 21 3 5 4 Protocol examples Multiphor II 22 3 5 5 Running a protocol sesssssssse 22 3 5 6 Troubleshooting ssssssssssse 23 3 6 IPGphor Isoelectric Focusing System 24 3 6 1 IPG strip rehydration IPGphor strip holder 24 3 6 2 Optional Apply electrode pads 24 3 6 3 Optional Apply sample after gel rehydration ssssssssssse 25 3 6 4 Isoelectric focusing guidelines 25 3 6 5 Protocol examples IPGphor 26 3 6 6 Running protocol sess 27 3 6 7 Troubleshooting ssssssssssse 27 Part Ill Second dimension SDS PAGE 4 0 Second dimension SDS PAGE overview 28 4 1 Background to SDS PAGE sssssse 28 4 2 IPG st
59. f the dye does not migrate no current 4 80007 7 30 60000 Step n hold is flowing If this occurs check the contact between the external face of the strip holder electrodes and the elec 13cm 1 rehydration 12 00 500 1 00 500 Step n hold trode areas on the instrument and between the rehy 3 1000 1 00 1000 Step n hold drated gel and the internal face of the electrodes s 8000 330 75000 Step n hold Note It is possible that the programmed maximum volt Ten i NN 1200 age will not be reached with the shorter IPG strips or 2 500 1 00 500 Step n hold with samples with high conductivity i HN n T E After IEF proceed to the second dimension separation 5 8000 230 20000 Step n hold immediately or store the IPG strips at 40 to 80 C in screw cap tubes The 7 cm strips fit in disposable 15 ml The total rehydration time can be adjusted somewhat for convenience but must be conical tubes 11 13 18 cm strips fit in 25 x 200 mm greater than 10 hours screw cap culture tubes This voltage may not be reached within the suggested step duration 26 USING IMMOBILIZED PH GRADIENTS PART II FIRST DIMENSION ISOELECTRIC FOCUSING 3 6 7 Troubleshooting Table 17 lists possible problems that could be encoun tered during IEF and how to solve them TABLE 17 TROUBLESHOOTING FIRST DIMENSION IEF IPGPHOR Symptom Possible cause Remedy Current too low or zero Electrical continuity is impeded Check the external electrode contacts Th
60. ge 35 Troubleshooting second dimension SDS PAGE Multiphor II flatbed system Symptom Possible cause Remedy No distinct spots are visible Sample is insufficient Increase the amount of sample applied Insufficient sample entered the IPG strip Increase the concentration of the solubilizing components in the sample due to poor sample solubilization solution See section 2 5 Composition of sample solution Sample contains impurities that Increase the focusing time or modify the sample preparation method See prevent focusing Part Sample Preparation The pH gradient is wrongly oriented The pointed end of the Immobiline DryStrip is the acidic end and should point toward the anode Flatbed gel format IPG strip is placed Ensure that the IPG strip is placed gel side down plastic backing upward s wrong side down on second dimension gel on the SDS second dimension gel ai i Detection method was not sensitive enough Use another detection method e g silver staining instead of Coomassie blue staining Failure of detection reagents Check expiration dates on staining solutions Prepare fresh staining solutions Individual proteins appear as multiple spots or Protein carbamylation Do not heat any solutions containing urea above 30 C as isocyanate a are missing unclear or in the wrong position urea degradation product will carbamylate proteins changing their pl Protein oxidation DTT in the rehydrati
61. hat no air bubbles are trapped between the IPG strip and the slab gel surface or between the gel backing and the glass plate Optional Apply molecular weight marker proteins The markers are applied to a paper IEF sample applica tion piece in a volume of 15 to 20 ul For less volume cut the sample application piece proportionally Place the IEF application piece on a glass plate and pipette the marker solution onto it then pick up the application piece with forceps and apply to the top surface of the gel next to one end of the IPG strip The markers should contain 200 to 1 000 ng of each component for Coomassie staining and about 10 to 50 ng of each component for silver staining Seal the IPG strip in place Imbedding the IPG strip in agarose prevents it from moving or floating in the electrophoresis buffer Prepare agarose sealing solution 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 minutes to fully melt the agarose Tip An ideal time to carry out this step is during IPG strip equilibration Allow the agarose to cool to 40 to 50 C and then slowly pipette the amount required to seal the IPG strip in place Figure 21 Pipetting slowly avoids introducing bubbles Allow a minimum of 1 minute for the agarose to cool and solidify 32 USING IMMOBILIZED PH GRADIENTS Figure 21 Finish assembling the electrophoresis unit
62. he 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 the surface of the solu tion tilting the strip holder slightly as needed to ensure complete and even wetting Figure 16 Figure 17 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 bubbles under the IPG strip Apply IPG 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 Q Place the cover on the strip holder Pressure blocks on the underside of the cover ensure that the IPG strip maintains good contact with the electrodes as the gel swells Q Allow the IPG strip to rehydrate Rehydration can proceed on the bench top or on the IPGphor unit platform Ensure that the holder is on a level surface A minimum of 10 hours is required for rehydration overnight is recommended The rehydra tion period can be programmed as the first step of an IPGphor protocol This is espe
63. he protein will partially remove SDS Precipitation at room temperature will maximize removal of SDS but protein precipitation is more complete at 20 C 43 Nucleic acids DNA RNA Nucleic acids increase sample viscosity and cause Treat samples rich in nucleic acids with a protease free 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 continues on following page 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 0 25 mg ml RNase A and 50 mM MgCl followed by incubation on ice 30 47 Note The DNase and RNase proteins 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 2 D ELECTROPHORESIS 11 PART l SAM
64. ia 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 elec trophoresis The sample can also be prefractionated by solubility under different extraction conditions prior to 2 D electrophoresis References 8 9 describe examples of this approach See reference 10 for an overview of the subject of protein fractionation Precipitation of the proteins in the sample and removal of interfering substances are optional steps The decision to employ these steps depends on the nature of the sample and the experimental goal Precipitation proce dures which are used both to concentrate the sample and to separate the proteins from potentially interfering substances are described in section 2 3 Removal tech niques which eliminate specific contaminants from the sample are described in section 2 4 as are the effects contaminants salts small ionic molecules ionic deter gents nucleic acids polysaccharides lipids and pheno lic 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 concen trations 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 rehy dration solution
65. ible In large studies with patterns containing several thousand spots it may be almost impossible to detect the appearance of a few new spots or the disappearance of single spots Image collection hardware and image evaluation software are necessary to detect these differences as well as to obtain maximum information from the gel patterns Amersham Pharmacia Biotech ImageMaster 2D Flite Software and 2D Database Software together with the Sharp JX 330 Scanner comprise a system that allows the user to capture store evaluate and present information contained in 2 D gels a The Sharp JX 330 Desktop Scanner captures optical information over a range from 0 to 3 0 OD from pixels as small as 42 um 600 dpi 1 ImageMaster 2D Elite Software provides the essential tools for analyzing complex protein samples separated by 2 D electrophoresis Protein spots are automatically detected 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 amount changes in spot patterns ImageMaster 2D Database Software adds a database search facility that searches and queries across experi ments and images and analyses experiments for quan titative pattern relationships 5 3 Standardization of results The 2 D electrophoresis technique is often used compar atively and thus requires a reproducible method for determining relative spot positions Bec
66. inched Ensure that an adequate level of buffer is in the upper reservoir Dye front is irregular Poor uneven polymerization of gel The top surface of the second dimension gel is not flat 4 4 Multiphor II flatbed system 4 4 1 ExcelGel preparation Two sizes of precast ExcelGel gradient SDS gels are avail able The 110 x 250 mm gel contains an 8 to 18 acry lamide gradient and the 180 x 250 mm gel contains a 12 to 14 acrylamide gradient Either gel accepts a single 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 comparative studies For maximum resolution the larger gel coupled with the 18 cm IPG strip is the best choice Important A flatbed second dimension system is not recommended if the first dimension has been run on a pH 6 11 IPG strip O Equilibrate the IPG strips Just prior to preparing the ExcelGel SDS gel equilibrate the IPG strips as described in section 4 2 2 Prepare the Multiphor II unit Set the temperature on the MultiTemp III Thermostatic Circulator to 15 C Pipette 2 5 to 3 0 ml of IPG Cover Fluid or kerosene onto the Multiphor II cooling plate Degas the gel solution or increase the amount of ammonium persulphate and TEMED by 50 Immediately after pouring the gel overlay the surface with water saturated butanol Place the ExcelGel SDS gel Remove the gel from the foil package by cutting away the edges of the pack
67. ion buffer Equilibration Place the IPG strips in individual tubes with the support film toward the tube wall screw cap culture tubes work well Add DTT containing equilibration solution to each tube Suggested volumes are 10 ml for 18 cm IPG strips 5 10 ml for 11 cm or 13 cm IPG strips and 2 5 5 ml for 7 cm IPG strips Cap the tube or seal it with flexible paraffin film and place it on its side on a rocker Equilibrate for 15 minutes Second equilibration recommended for flatbed second dimension optional for vertical second dimension A second equilibration may be performed with an iodoacetamide solution without DTT Prepare a solu tion of 250 mg iodoacetamide per 10 ml SDS equilibra tion buffer Note This second equilibration step reduces point streaking and other artifacts when using a flatbed system for the second dimension SECOND DIMENSION SDS PAGE Decant the first equilibration solution and add iodoac etamide containing equilibration solution to each tube Suggested volumes are 10 ml for 18 cm IPG strips 5 10 ml for 11 cm or 13 cm IPG strips and 2 5 5 ml for 7 cm IPG strips Cap the tube or seal it with flexible paraffin film place it on its side on a rocker and equilibrate for 15 minutes O 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 equi libration solution place the I
68. lectrophoresis Electrophoresis 19 837 844 1998 10 Deutscher M P ed Guide to Protein Purification Metbods Enzymol 182 1 894 1990 44 USING IMMOBILIZED PH GRADIENTS 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Dunn M J Corbett J M 2 dimensional polyacry lamide gel electrophoresis Methods Enzymol 271 177 203 1996 Rabilloud T Solubilization of proteins for elec trophoretic analysis Electropboresis 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 Electropboresis 18 307 316 1997 Bollag D M Edelstein S J Protein Metbods Chapter 2 Protein Extraction Wiley Liss 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 dimen sional polyacrylamide gel electrophoresis and image processing Electrophoresis 15 984 987 1994 Sanchez J C Appel R D Golaz O Pasquali C Ravier E Bairoch A Hochstrasser D F Inside SWISS 2DPAGE database Electrophoresis 16 1131 1151 1995
69. lin 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 one 37 38 39 40 41 42 43 44 45 46 47 48 North M J Prevention 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 elec trophoresis Applications for cell signaling in normal and cancer cell
70. lting 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 of removing salts and other contaminants but can also result in losses see section 2 3 Endogenous small ionic molecules Endogenous small ionic molecules are present in any cell lysate TCA acetone precipitation is particularly effective at removing this nucleotides metabolites These substances are often negatively charged and can result in sort of contaminant Other desalting techniques may be applied phospholipids etc poor focusing toward the anode see above lonic detergent lonic detergent usually SDS is often used during protein extraction Dilute the SDS containing sample into a rehydration solution and solubilization but can strongly interfere with IEF SDS forms complexes with proteins and the resulting negatively charged complex will not properly focus unless the SDS is removed or 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 24 sequestered Acetone precipitation of t
71. ly 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 Buffer strip slides out Incorrect electrode placement Ensure that the electrodes are aligned over the centre of the buffer strips before from under electrode lowering the electrode holder 2 D ELECTROPHORESIS 35 PART IV VISUALIZATION Part IV Visualization and analysis of results 5 0 Visualization of results Most detection methods used for SDS gels can be applied to second dimension gels Autoradiography and fluorography are the most sensi tive detection methods To employ these techniques the sample must consist of protein radiolabeled in vivo using either 5S C H or in the case of phosphoproteins P For autoradiographic detection the gel is simply dried and exposed to X ray film or a storage phosphor screen Fluorography is a technique that provides extra sensitiv ity by impregnating the gel in a scintillant such as PPO 2 4 diphenyloxazole prior to drying Silver staining is the most sensitive nonradioactive method Silver staining is a complex multistep process and many variables can influence the results High purity reagents and precise timing are necessary for reproducible high quality results Impurities in the gel and or the water used for preparing the staining reagents can give poor staining results Coomassie staining although 50 fold less
72. mated Gel Stainer with 19 x 29 cm PTFE coated stainless steel tray Recommended for 125 200 ml volume Hoefer Automated Gel Stainer with 29 x 35 cm PTFE coated stainless steel tray Recommended for 250 400 ml volume Coomassie tablets PhastGel Blue R 350 Protocol Guide Hoefer Automated Gel Stainer Hoefer SE 1200 Easy Breeze Air Gel Drier 115 V Hoefer SE 1200 Easy Breeze Air Gel Drier 230 V Hoefer GD 2000 Vacuum Gel Drying System 115 V Hoefer GD 2000 Vacuum Gel Drying System 230 V ImageMaster Image Analysis System 80 6350 56 80 6351 13 18 1108 33 18 1108 95 ImageMaster 2D Elite Software ImageMaster 2D Database Software Sharp JX 330 110 V Scanner A4 with transmission film scanning option I F SCSI 2 cable and Photoshop plug in software Sharp JX 330 220 V Scanner A4 with transmission film scanning option I F SCSI 2 cable and Photoshop plug in software 50 USING IMMOBILIZED PH GRADIENTS Asia Pacific Tel 852 2811 8693 Fax 852 2811 5251 Australasia Tel 61 2 9894 5188 Fax 61 2 9899 7511 Austria Tel 01 576 0616 10 Fax 01 576 0616 27 Belgium Tel 0800 73888 Fax 03 272 16 37 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 Central Europe Tel 43 1 982 3826 Fax 43 1 985 8327 Denmark Tel 45 16 24 00 Fax 45 16 24 24 Finland Tel 09 512 3940 Fax 09 512 1710 Former Soviet Union Tel 7 095 232 0250 Fax 7 095 232 6377 France Amersham products Tel 0169 18 28 00 Fax 0169 29 00 52 Pharma
73. n protocol Up to 12 strip holders can be used 3 6 1 IPG strip rehydration IPGphor strip holder O Prepare the strip holder s Select the strip holder s corresponding to the IPG strip length chosen for the experiment Important Handle the ceramic holders with care as they are brittle Wash each holder with detergent to remove residual protein Rinse thoroughly with double distilled water Use a cotton swab or a lint free tissue to dry the holder or allow it to air dry Handle clean holders with gloves to avoid contamination Note The holder must be completely dry before use Apply the rehydration solution Figure 15 Pipette the appropriate vol ume of rehydration solution into each holder as indicated in Table 15 Deliver the solu tion slowly at a central point in the strip holder channel away from the sample appli cation wells Remove any larger bubbles Figure 15 Important To ensure complete sample uptake do not apply excess rehydration solution TABLE 15 REHYDRATION SOLUTION VOLUME PER IPG STRIP IPG strip length cm Total volume per strip ul 1 125 n m 18 350 Including sample if applied 24 USING IMMOBILIZED PH GRADIENTS FIRST DIMENSION ISOELECTRIC FOCUSING Place the IPG strip Figures 16 and 17 gg Remove the protective cover from the IPG strip Position the IPG strip with the gel side down and the pointed anodic end of the strip directed toward t
74. n when the same stock solution will be used with different pH range IPG strips See section 3 4 2 Tracking dye bromophenol blue provides a monitor for IEF progress at the beginning of the protocol If the tracking dye does not migrate toward the anode no current is flowing Note however that the dye leaves the strip well before the sample is focused Sample can be applied by including it in the rehydra tion solution Up to 1 mg of sample per strip can be diluted into or redissolved in rehydration solution just prior to IEF The amount of sample required is TABLE 9 ADDITION OF IPG BUFFER TO THE REHYDRATION SOLUTION Suggested carrier ampholytes IEF system pH range of IPG strip for rehydration solution Recommended concentration Multiphor II 4 7 L 3 10 L or 3 10 NL IPG Buffer with pH range 2 IPG Buffer 50 ul per 2 5 ml identical to that of IPG strip Multiphor II 6 11 L pH 6 11 L IPG Buffer 0 5 IPG Buffer 12 5 ul per 2 5 ml IPGphor 4 7 L 3 10 L 3 10 NL or 6 11 L IPG Buffer with pH range identical 0 5 IPG Buffer 12 5 ul per 2 5 ml to that of IPG strip 2 D ELECTROPHORESIS 17 PART II dictated in part by the detection or visualization method used Radiolabeling requires a very small amount of sample silver staining requires typically 1 to 100 pg of sample and Coomassie blue staining and preparative applications require larger amounts 3 4 2 Rehydration solution preparation Prepare the rehydration st
75. 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 24 31 55 Reducing agents are frequently included in the sample solution to break any disulphide 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 Dithioery thritol DTE is similar to DTT and can also be used as a reducing agent Originally 2 mercaptoethanol was used as a reductant but higher concentrations of the reductant are required and inherent impurities may result in arti facts 56 More recently the non thiol reductant tributyl phosphine at a concentration of 2 mM has been used as a reductant for 2 D samples 57 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 A sample should remain in sample solution at room temperature for at least 1 hour 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 as heating in the pres ence of ure
76. 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 PART IIl lodoacetamide alkylates thiol groups on proteins preventing their reoxidation during electrophoresis Protein reoxidation during electrophoresis can result in streaking and other artifacts Iodoacetamide also alkylates residual DTT to prevent point streaking and other silver staining artifacts 65 Iodoacetamide 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 staining Equilibration with iodoacetamide is also used to minimize unwanted reactions of cysteine residues i e when mass spec troscopy is to be performed on the separated proteins p Tracking dye bromophenol blue allows monitoring of electrophoresis 4 2 2 Equilibration steps Note The second dimension gel must be ready for use prior to IPG strip equilibration See sections 4 3 1 and 4 4 1 for preparation of vertical and horizontal gels respectively 9 Prepare equilibration solution Prepare SDS equilibration buffer see Appendix solu tion D This is a stock solution Just prior to use add 100 mg DTT per 10 ml SDS equilibrat
77. nic detergents can be used instead of CHAPS 42 USING IMMOBILIZED PH GRADIENTS C Rehydration stock solution with IPG Buffer 8 M urea 2 CHAPS 0 5 or 2 IPG Buffer bromophenol blue 25 ml Final concentration Amount Urea FW 60 06 8M 12g same pH range as the IPG strip Store in 2 5 ml aliquots at 20 C DIT is added just prior to use 7 mg DIT per 2 5 ml aliquot of rehydration stock solution If loading sample by inclusion in the rehydration solution sample is also added to the 2 5 ml aliquot of rehydration solution just prior to use Either of two IPG Buffer concentrations is recommended depending on the IEF system used and e pH range of the IPG strip Refer to Table 9 3 f 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 sed instead of CHAPS Selection of IPG Buffer concentration is based on IEF system used and pH range of the IPG strip Refer to Table 9 5 Use 125 ul IPG Buffer for a 0 5 concentration and 500 ul IPG Buffer for a 2 concentration D SDS equilibration buffer 50 mM Tris Cl pH 8 8 6 M urea 30 glycerol 2 SDS bromophenol blue 200 ml Final concentration Amount 1 5 M Tris Cl pH 8 8 see solution P S0mM 8 Urea FW 60 06 6M 72 07 g Glycerol 87 vv 30 v v 69 ml SDS FW 288 38 QW 40g Bromophenol blue
78. nsion gel before applying buffer strips and IPG strip Flatbed gel format IPG strip not removed Remove the IPG strip and application pieces from the second dimension during electrophoresis gel when the bromophenol blue dye from has moved away from the IPG strip by 4 6 mm Flatbed gel format Air bubbles under the Ensure that no bubbles are trapped under the second dimension gel during second dimension gel cause uneven placement on the cooling plate migration due to poor heat transfer Flatbed gel format Water drops or pieces Take care that nothing is dropped or splashed onto the surface of the of buffer strip on the surface of the second second dimension gel dimension gel Horizontal streaking or incompletely focused spots zx Sample not completely solubilized prior Be sure that the sample is completely and stably solubilized 7 to application Note Repeated precipitation resolubilization cycles produce or increase horizontal streaking See section 2 5 Composition of the sample solution for general guidelines for sample solubilization Sample is poorly soluble in rehydration Increase the concentration of the solubilizing components in the rehydration solution solution See section 3 4 IPG strip rehydration solution Increase concentration of IPG Buffer 38 USING IMMOBILIZED PH GRADIENTS TROUBLESHOOTING TABLE 26 TROUBLESHOOTING 2 D RESULTS continued Symptom Horizontal streaking or incompletel
79. ntly 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 tech nique Which is capable of simultaneously resolving thousands of proteins in one separation procedure The replacement of classical first dimension carrier ampholyte pH gradi ents 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 electrophore sis 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 instru ments for IPG IEF 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 immobilized pH gradients for the first dimension it provides de
80. ock solution Recommended formulations are listed in the Appendix solutions B and C Select the formulation appropriate to the experiment Note Stock solution can be stored in 2 5 ml aliquots at 20 C Just prior to use slowly thaw a 2 5 ml aliquot of stock solution Add the appropriate amount of IPG Buffer if it is not already included in the rehydration stock solution Refer to Table 9 9 Add 7 mg DTT and sample if desired Note DTT and the sample must be added fresh just prior to use 3 5 Multiphor ll and Immobiline DryStrip Kit 3 5 1 IPG strip rehydration Immobiline DryStrip Reswelling Tray The Immobiline DryStrip Reswelling Tray has 12 inde pendent reservoir slots that can each hold a single IPG strip up to 18 cm long Separate slots allow the rehydration of individual IPG strips in a minimal volume of solution O Prepare the Reswelling Tray Figure 9 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 Sey ES centred Ensure that the JJ tray is clean and dry Figure 9 O Apply the rehydration solution Pipette the appropriate volume of rehydration solution into each slot as indicated in Table 10 Deliver the solution slowly to the center of the slot Remove any large bubbles Important To ensure complete sample uptake do not apply excess rehydration solution 18 USING IMMOBILIZED PH GRADIENTS
81. of polypeptides in the size range below 10 kDa ExcelGel precast gels for second dimension SDS PAGE on the Multiphor II flatbed system utilize a different tris tricine buffer system 4 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 optional equilibration step replaces the reductant with iodoacetamide 4 2 1 Equilibration solution components Equilibration introduces reagents essential for the second dimension separation gt Equilibration buffer 50 mM Tris HCl pH 8 8 main tains IPG strip pH in a range appropriate for electrophoresis gt Urea 6 M together with glycerol reduces the effects of electroendosmosis by increasing the viscosity of the buffer 3 Electroendosmosis is due to the presence of fixed charges on the IPG strip in the electric field and can interfere with protein transfer from the IPG strip to the second dimension gel gt Glycerol 30 together with urea reduces elec troendosmosis and improves transfer of protein from the first to the second dimension 3 DTT preserves the fully reduced state of denatured unalkylated proteins Sodium dodecyl sulphate SDS denatures proteins and forms negatively charged protein SDS complexes The amount of SDS bound to a protein and therefore the additional
82. often occur when loading with sample cups 1 This method is technically simpler avoiding problems of leakage that can occur when using sample cups There are however cases when one might prefer to load the sample following rehydration immediately prior to IEF If proteolysis or other protein modifications are a concern overnight rehydration with sample may not be desired 1 Better results are often obtained on pH 6 11 L IPG strips when the sample is loaded anodically in a sample cup or sample well Guidelines for sample application after rehydration using the Multiphor II and Immobiline DryStrip Kit system are given in section 3 5 2 D 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 IPGphor system guidelines for sample application after rehydration are given in section 3 6 3 Sample is pipetted into sample application wells located at each end of the strip holder Up to 7 5 pl of sample solution can be added to each side i e 15 pnl per well or 30 pl total if both sides of both wells are used PART II 3 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 the Multiphor II system is used for IEF or in IPGphor strip holders if the IPGphor is used Rehydration solution which may or may not include the sample is
83. on and equilibration solutions keeps the disulphide bonds reduced For additional protection include an iodoacetamide treatment during equilibration prior to the second dimension separation lodoacetamide alkylates the thiol groups to prevent the reduced proteins from reoxidizing Spots are vertically doubled or twinned Vertical gel format IPG strip is not Ensure that the plastic backing of the IPG strip is against the glass plate LI placed properly on the second dimension gel LJ b as 6 j u 2 D ELECTROPHORESIS 37 TROUBLESHOOTING TABLE 26 TROUBLESHOOTING 2 D RESULTS continued Symptom Possible cause Remedy Distortion of 2 D pattern LJ m n o si Vertical gel format The top surface of the Immediately after pouring the gel overlay the surface with second dimension gel is not flat water saturated butanol O wo i di amp E Vertical gel format Uneven polymerization Degas the gel solution of gel due to incomplete polymerization too rapid polymerization or leakage during gel casting Polymerization can be accelerated by increasing by 50 the amount of ammonium persulphate and TEMED used Polymerization can be slowed by decreasing by 33 the amount of ammonium persulphate and TEMED used Ensure that there is no leakage during gel casting Flatbed gel format Moisture on the surface Allow ExcelGel to dry for about 5 minutes after removing plastic cover and of the second dime
84. ons e g sample and rehydration solution composition IPG strip length and pH gradient will require an empirical determination for optimal results An approximate time is given in the example protocols provided in Table 13 One factor that increases required focusing time is the presence of small ions which must move to the ends of the IPG strips before protein focusing can occur Larger quantities of protein also require more time to focus Note Over focusing is seldom a problem below 100 000 total volt hours but on longer runs it may contribute to horizontal streaking visible in the 2 D result See also section 6 0 Troubleshooting 2 D results 2 D ELECTROPHORESIS 21 PART II 3 5 4 Protocol examples Multiphor Il The protocols given in Table 13 are suitable for first dimension isoelectric focusing of protein samples in typi cal analytical quantities with IPG Buffer concentrations of 0 5 to 2 in the rehydration solution Optimal focus ing 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 up to 100 000 volt hours Vh if necessary Note Sample application onto pH 6 11 L IPG strips by inclusion in the rehydration solution significantly prolongs the time required for complete focusing FIRST DIMENSION ISOELECTRIC FOCUSING Increase the recommended volt h
85. ons for subsequent second dimension electrophoresis of IPG strips Part IV discusses visualization and analysis of the 2 D electrophoresis results The 2 D protocols described herein use products of Amersham Pharmacia Biotech Equipment choices are discussed in section 1 2 1 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 accord ing to their molecular weights MW 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 Under the influence of an electric current carrier ampholyte
86. or 2 D electrophoresis and is more effective than either TCA or acetone alone Suspend lysed or disrupted sample in 10 TCA in acetone with either 0 07 2 mercaptoethanol or 20 mM DTT Precipitate proteins for at least 45 minutes 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 4 25 31 40 48 49 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 Precipitation with ammonium acetate in methanol following phenol extraction This technique has proven useful with plant samples containing high levels of interfering substances 10 USING IMMOBILIZED 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 ammonium acetate in methanol and then with acetone Residual acetone is evaporated 40 39 44 50 PH GRADIENTS The method is complicated and time consuming PART l SAMPLE PREPARATION 2 4 Removal of contaminants that affect 2 D results Non protein impurities in the sample can interfere with separation and subsequent visualization of the 2 D result so sample preparation can include steps to rid the sample of these su
87. or Coomassie staining and about 10 to 50 ng of each component for silver staining Q Position electrodes Figure 25 Place the IEF electrode holder on the electrophoresis unit in the upper position and align the electrodes with the centre of the buffer strips Plug in the electrode connectors and carefully lower the electrode holder onto the buffer strips Check that the buffer strips have not moved 4 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 24 Note It is important to use a protocol with a low current sample entry phase Remove the IPG strip and application pieces and move the cathodic buffer strip prior to the second higher current phase as indicated in footnote 1 of Table 24 TABLE 24 ELECTROPHORESIS CONDITIONS FOR EXCELGEL Voltage Current Power Duration Step V mA W h min ExcelGel SDS gradient 8 18 1 600 20 30 0 25 0 30 2 600 50 30 1 10 ExcelGel XL SDS gradient 12 14 1 1000 20 40 0 45 2 1000 40 40 2 40 When the bromophenol blue dye front has moved away from the IPG strip by 4 6 mm for ExcelGel XL SDS 12 14 or by 1 2 mm for ExcelGel SDS 8 18 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 Electrophoresis is stopped 5 minutes afte
88. ours in the final phase of the program by 6 to 10 fold for 7 cm long IPG strips 5 to 8 fold for 11 cm long IPG strips and 5 to 7 fold for 13 and 18 cm long IPG strips 3 5 5 Running a protocol Ensure that the electrodes on the Immobiline DryStrip tray are connected and place the lid on the Multiphor II unit Connect the leads on the lid to the power supply Ensure that the current check on the EPS 3501 XL power supply is switched off Begin IEF As isoelectric focusing proceeds the bromophenol blue tracking dye migrates toward the anode Note that the TABLE 13 IMMOBILINE DRYSTRIP IEF GUIDELINES FOR MULTIPHOR II Program EPS 3501 XL power supply in gradient mode with current check option turned off IPG strip is rehydrated with a solution containing IPG Buffer of the corresponding pH range Immobiline DryStrip Phase Voltage V Current mA Power W Duration h min Vh recommended length pH range s 7cm pH 4 71 1 200 2 5 0 01 1 2 3500 2 b 1 30 2800 3 3500 2 5 0 55 1 30 3200 5200 Total 2 25 3 00 6000 8000 7cm pH 6 11 17 pH 3 10 L and pH 3 10 NL 1 200 2 5 0 01 1 2 3500 2 5 1 30 2800 3 3500 2 5 0 35 1 05 2200 3700 Total 2 05 2 35 5000 6500 11 cm pH 4 71 1 300 2 5 0 01 1 2 3500 2 5 1 30 2900 3 3500 2 5 2 20 3 30 8100 12100 Total 3 50 5 00 11000 15000 11 cm pH 6 11 L and pH 3 10 L 1 300 2 5 0 01 1 2 3500 2 5 1 30 2900 3 3500 2 5 1 45 2 35 6100 9100 Total 3 15 4 05 9000 12000 13 cm pH 4 71 1 300 2 5
89. pared incorrectly E TI Insufficient SDS in SDS electrophoresis Use 0 1 w v SDS E 4 buffer T Vertical gap in 2 D pattern Tea Wi a Modify sample preparation See section 2 4 Removal of contaminants that affect 2 D results Impurities in sample Impurities in rehydration solution components 40 USING IMMOBILIZED PH GRADIENTS Use only high quality reagents De ionize urea solutions TROUBLESHOOTING TABLE 26 TROUBLESHOOTING 2 D RESULTS continued Symptom Possible cause 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 Remedy 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 the second dimension gel 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 Vertical regions of poor focusing ik x rf B amd all rM The IPG strip was not fully rehydrated 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
90. proteins are naturally found in complexes with membranes nucleic acids or other proteins some proteins form vari ous non specific aggregates and some proteins precipi tate when removed from their normal environment The effectiveness of solubilization depends on the choice of cell disruption method protein concentration and disso lution method choice of detergents and composition of the sample solution If any of these steps is not optimized for a particular 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 frac tion Both gentle and vigorous lysis methods are discussed in section 2 1 6 USING IMMOBILIZED PH GRADIENTS SAMPLE PREPARATION Proteases may be liberated upon cell disruption Proteol ysis 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 2 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 mitochondr
91. r DALT see User Manual 30 USING IMMOBILIZED PH GRADIENTS SECOND DIMENSION SDS PAGE b Calculate the formulation of the gel solution The recipes given in Table 20 produce 100 ml of solution for a single percentage gel The recipes in Table 21 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 c Prepare the gel solution in a vacuum flask omitting the TEMED and ammonium persulphate Add a small magnetic stir bar Stopper the flask and apply a vacuum for several minutes while stirring on a magnetic stirrer Add the TEMED and ammonium persulphate and gently swirl the flask to mix being careful not to generate bubbles Immediately pour the gel Pour and prepare the gel Fill the gel cassette to 3 to 10 mm below the top No stacking gel layer is required Overlay each gel with a thin layer 100 to 500 pl of water saturated n i or t butanol immediately after pouring to minimize gel exposure to oxygen and to create a flat gel surface After allowing a minimum of 1 hour for polymerization inspect each gel and ensure that polymerization is even and complete and that the top surface of each gel is straight and flat Remove the overlay and rinse the gel surface with gel storage solution see Appendix solution I Storage of unused gels Gels not used immediately can be stored for future use at 4 C for up to two weeks Gel
92. r User Manual for 3 1000 100 1000 Sisp ho instructions on how to program a protocol 4 8000 2 00 16000 Step n hold 3 6 6 Running a protocol 13cm 1 rehydration 12 00 Ee 7 500 10 500 Step n hold Ensure that the strip holders are properly positioned on 3 1000 1 00 1000 Step n hold the IPGphor platform Use the guidemarks along the 4 8000 2 00 16000 Step n hold sides of the platform to position each strip holder and OPO Io mm check that the pointed end of the strip holder is over the 2 500 100 500 Step n hold anode pointing to the back of the unit and the blunt 3 1000 1 00 1000 Step n hold end is over the cathode Please refer to the IPGphor User 4 8000 4 00 32000 Step n hold Manual Check that both external electrode contacts on the underside of each strip holder make metal to metal pH gradient 6 11 L contact with the platform s Close the safety lid At least two of the three pressure Step Voltage EJ ea oso pads under the safety lid must press gently against the cover of each strip holder to ensure contact between the Tem in son mem electrodes and the electrode areas Begin IEF a 1000 0 30 500 i As isoelectric focusing proceeds the bromophenol blue 4 8000 345 30000 Step n hold tracking dye migrates toward the anode Note that the dye front leaves the IPG strip well before focusing is JEJE A Pd Ke LNU l learing of the dye is no indication that the 500 1 00 500 Step n hold TIE Ey 3 1000 1 00 1000 Step n hold sample is focused I
93. r the bromophenol blue front has just reached the anodic buffer strip Remove and discard the buffer strips Figure 22 Figure 23 34 USING IMMOBILIZED PH GRADIENTS Figure 24 Figure 25 PART III SECOND DIMENSION SDS PAGE 4 4 4 Troubleshooting Table 25 lists possible problems that could be encoun tered during second dimension SDS PAGE using the Multiphor II flatbed system and how to solve them TABLE 25 TROUBLESHOOTING SECOND DIMENSION SDS PAGE MULTIPHOR Il FLATBED SYSTEM Symptom Possible cause Remedy No current at start of run The electrode cable is not plugged in Ensure that all cables are properly connected Dye front curves up Cathodic buffer strip does not contact the gel at Ensure that the cathodic buffer strip is centred and covers the entire width of the smiles at one edge the one edge second dimension gel Dye front curves up Inadequate cooling Ensure that the thermostatic circulator is connected to the Multiphor Il unit and smiles at both edges functioning correctly Dye front is irregular Some dye front irregularity results from the use of IPG Buffer and does not affect results Buffer strips or ExcelGel are old Ensure that the expiration dates on the buffer strips and ExcelGel have not elapsed Bubbles under the buffer strip Ensure that the buffer strips are placed firmly on the gel with no air bubbles trapped beneath them Bubbles under the IPG strip Ensure that the IPG strip is placed firm
94. re used Accessory divider plates increase the capacity to four gels 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 system Figure 3 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 possi bility 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 the Hoefer miniVE Figure 4 or the SE 260 are recommended The second dimension separation is typically complete in 1 to 2 hours 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 Figure 5 is recom mended The SE 600 accommodates up
95. reductants during tissue extraction e g DTT 2 mercaptoethanol sulphite 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 poor focusing Insoluble material in the sample can clog gel pores and result in Samples should always be clarified by centrifugation prior to application to first dimension IEF Insoluble material is particularly problematic when the sample is applied using sample cups It can prevent protein entry into the IPG strip 2 5 Composition of sample solution In order to achieve a well focused first dimension sepa ration sample proteins must be completely disaggre gated and fully solubilized Regardless of whether the sample is a relatively crude lysate or additional sample precipitation steps have been employed the sample solu tion must contain certain components to ensure complete solubilization and denaturation prior to first dimension IEF These always include urea and one or more detergents The lysis solution in the Appendix solution A containing urea and the zwitterionic deter gent CHAPS has been found to be effective for solubi lizing a wide range of samples Reductant and IPG Buffer are also frequently added to the sample solution
96. representation is desired This method can however be used for prefractionation or enrichment Residual ammonium sulphate will interfere with IEF and must be removed 42 See section 2 4 on removal of salts TCA precipitation TCA trichloroacetic acid is a very effective protein precipitant TCA is added to the extract to a final concentration of 10 20 and the proteins are allowed to precipitate on ice for 30 minutes 43 Alternatively tissue may be homogenized directly into 10 20 TCA 32 44 This approach limits proteolysis and other protein modifications Centrifuge and wash pellet with acetone or ethanol to remove residual TCA Proteins may be difficult to resolubilize and may not resolubilize completely Residual TCA must be removed by extensive washing with acetone or ethanol Extended exposure to this low pH solution may cause some protein degradation or modification Acetone precipitation This organic solvent is commonly used to precipitate proteins Many organic soluble contaminants e g detergents lipids will remain in solution Add at least 3 volumes of ice cold acetone to the extract Allow proteins to precipitate at 20 C for at least 2 hours Pellet proteins by centrifugation 43 4547 Residual acetone is removed by air drying or lyophilization Precipitation with TCA in acetone The combination of TCA and acetone is commonly used to precipitate proteins during sample preparation f
97. rip equilibration sssesssssse 28 4 2 1 Equilibration solution components 28 4 2 2 Equilibration steps 29 4 3 Vertical systems ssssseeeeeee 29 4 3 1 Preparing SDS slab gels vertical systems sse 29 4 3 2 Applying the equilibrated IPG strip 32 4 3 3 Electrophoresis conditions 32 4 3 4 Troubleshooting sss sese 33 4 4 Multiphor II flatbed system 33 4 4 1 ExcelGel preparation 33 4 4 2 Applying the equilibrated IPG strip 34 4 4 3 Electrophoresis conditions 34 4 4 4 Troubleshooting soseo 35 Part IV Visualization and analysis of results 5 0 Visualization of results ssssessssssss 36 5 1 Blotting us se PTS FERA AIS 36 5 2 Evaluation eR 36 5 3 Standardization of results ssssusss 36 Troubleshooting 6 0 Troubleshooting 2 D results 37 Appendix Solutions 42 References o 44 Ordering information 47 INTRODUCTION Introduction 1 0 Introduction to the manual This manual is divided into four parts Part I provides guidelines for sample preparation Part II details proce dures for performing the first dimension of 2 D elec trophoresis Part III contains general directi
98. rip gels are available from Amersham Pharmacia Biotech with the pH gradients 4 7 L linear 6 11 L linear 3 10 L linear and 3 10 NL non linear Available strip lengths are 7 11 13 and 18 cm The pH 3 10 L IPG strips have a linear pH gradient between pH 3 and pH 10 The pH 3 10 NL IPG strips have a roughly sigmoidal gradient that gives improved resolution between pH 5 and pH 7 16 USING IMMOBILIZED PH GRADIENTS FIRST DIMENSION ISOELECTRIC FOCUSING If a specialized pH gradient is required recipes for preparing custom narrow and wide immobilized pH gradients are given in 62 A pH 3 10 IPG strip will display the widest range of proteins on a single 2 D gel The narrower pH ranges are used for higher resolution separations in a particu lar pH range 3 3 Sample application method selection Sample can be applied either by including it in the rehy dration solution or by applying it directly to the rehydrated IPG strip via sample cups or sample wells It is usually preferable to load the sample onto the IPG strip by including the sample in the rehydration solution see section 3 4 Advantages to this mode of application include the following 1 This method allows larger quantities of protein to be loaded and separated 60 61 1 This method allows more dilute samples to be loaded Because there is no discrete application point this method eliminates the formation of precipitates at the application point that
99. rmance 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 IEF IEF is performed with the IPG strips placed horizontally on a flatbed electrophoresis unit Advantages to using the flatbed format include the following 1 soelectric 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 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 2 D ELECTROPHORESIS 15 PART II A Gorg et al 3 4 pioneered the development and use of IPG strips for the first dimension of 2 D electrophoresis The protocols presented in this manual are largely based on the work of A Gorg and her colleagues The IPG strips are rehydrated in a solution containing the necessary additives and optionally the sample proteins The rehy dration solution is described in detail in section 3 4 IEF is performed
100. rt Sample preparation 906000000009090000090000900000900000900000900000900000909000099000999 2 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 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 solu bilize different types of protein samples some
101. s Electrophoresis 15 417 440 1994 Meyer Y Grosset J Chartier Y Cleyet Marel J C Preparation by two dimensional electrophore sis of proteins for antibody production Antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplasts Elec trophoresis 9 704 712 1988 Halloway P Arundel P High resolution two dimensional electrophoreisis of plant proteins Anal Biochem 172 8 15 1988 Flengsrud R Kobro G A method for two dimen sional 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 2 D ELECTROPHORESIS 45 49 50 51 52 53 54 55 56 57 58 46 REFERENCES 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 d
102. s blocks the current and physically prevents the protein from entering the IPG strip The ionic strength of the sample is higher than that of the 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 Movement may stop all together Replace IPG strip and reapply sample cup Dilute the sample as much as possible or just prior to loading dialyse the sample to remove salts Sparking or burning of IPG strips Conductivity of the sample IPG strips is too high Ensure that the sample is adequately desalted Or before raising the voltage to maximum include a prolonged low voltage phase in the IEF protocol to allow the ions to move to the ends of the IPG strip 2 D ELECTROPHORESIS 23 PART II 3 6 IPGphor Isoelectric Focusing System With the IPGphor Isoelectric Focusing System both rehy dration of the IPG strip and IEF occur in individual strip holders Different length holders are available for the different length IPG strips A strip holder is made of ther mally conductive ceramic with built in platinum elec trodes and a transparent lid The sample can be loaded by simply including it in the rehydration solution or the sample can be loaded separately just prior to IEE Once sample is applied to the IPG strip and the strip holder is in place on the IPGphor unit platform the remaining steps are carried out automatically according to the chose
103. s form a pH gradient a critical component of IEF See section 3 1 Background to IEF for more detail Sample was applied to one end of each tube gel and separated at high voltages After IEF the gel rods were removed from their tubes equilibrated in SDS sample buffer and placed on vertical SDS polyacrylamide gels for the second dimension separation The power of 2 D electrophoresis as a biochemical sepa ration technique has been recognized virtually since its introduction Its application however has become signif icant only in the past few years as a result of a number of developments 1 The 2 D technique has been improved to generate 2 D maps that are superior in terms of resolution and repro ducibility This new 2 D technique developed by A Gorg and colleagues 3 4 utilizes an improved first dimension separation method that replaces the carrier ampholyte generated pH gradients with immobilized pH gradients IPG and replaces the tube gels with gel strips supported by a plastic film backing A more detailed discussion of the merits of this technique is presented in section 3 1 Background to IEF 1 Methods for the rapid analysis of proteins have been improved to the point that single spots eluted or trans ferred from single 2 D gels can be rapidly identified Mass spectroscopic techniques have been developed that allow analysis of very small quantities of peptides and proteins Chemical microsequencing and amino aci
104. search purity checks and microscale protein purification This manual describes methods for 2 D electrophoresis using precast IPG strips Immobiline DryStrip gels available from Amersham Pharmacia Biotech The 2 D process begins with sample preparation Proper sample 2 D ELECTROPHORESIS 1 INTRODUCTION TABLE 1 EQUIPMENT CHOICES FOR 2 D ELECTROPHORESIS Choices for first dimension IEF Multiphor ll Electrophoresis unit with Immobiline DryStrip Kit Rehydration in Reswelling Tray IEF in Multiphor II unit with Immobiline DryStrip Kit Choice Factors Figure 1 Multipbor II Electrophoresis unit a Multiphor II can be used for both first and second with Immobiline DryStrip Kit dimension separations Multiphor II is a versatile system Its use is not limited to IEF with IPG strips Several different electrophoresis techniques can be performed with the instrument IPGphor Isoelectric Focusing System Rehydration and IEF both in IPGphor strip holder Choice Factors Rehydration and IEF can be performed overnight unattended 1 Fewer IPG strip manipulations are required reducing the chance of error a Faster separations and sharper focusing are possible because of higher voltage Power supply and temperature control are built into Figure 2 IPGpbor Isoelectric Focusing System the instrument 2 USING IMMOBILIZED PH GRADIENTS INTRODUCTION TABLE 1 EQUIPMENT CHOICES FOR 2 D ELECTROPHORESIS
105. sues 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 General procedure Sonicate cell suspension in short bursts to avoid heating Cool on ice between bursts French pressure cell 20 21 24 Cells are lysed by shear forces resulting from forcing cell suspension through a small orifice under high pressure Microorganisms with cell walls bacteria algae yeasts Place cell suspension in chilled French pressure cell Apply pressure and collect extruded lysate Grinding 4 7 25 26 Some cell types can be opened by hand grinding with a mortar and pestle Solid tissues microorganisms Tissue or cells are normally frozen with liquid nitrogen and ground to a fine powder Alumina or sand may aid grinding Mechanical homogenization 8 16 27 29 Many different devices can be used to mechanically homogenize tissues Handheld devices such as Dounce or Potter Elvehjem homogenizers can be used 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 Solid tissues Chop tissue into small pieces if necessary Add chilled homogenization buffer 3 5 volumes to volume of tissue Homogenize briefly Clarify lysate
106. t B Ek K Righetti P G Gianazza E Gorg A Westermeier R Postel W Isoelectric focusing in immobilized pH gradients principle methodology and some applications J Biochem Biophys Methods 6 317 339 1982 USING IMMOBILIZED PH GRADIENTS 59 60 61 62 63 64 65 Bjellqvist B Sanchez J C Pasquali C Ravier F Paquet N Frutiger S Hughes G J Hochstrasser D Micropreparative two dimensional electrophore sis 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 immobi lized pH gradients Electrophoresis 18 324 327 1997 Rabilloud T Valette C Lawrence J J Sample application by in gel rehydration improves the reso lution of two dimensional electrophoresis with immobilized pH gradients in the first dimension Electrophoresis 15 1552 1558 1994 Westermeier R Electrophoresis In Practice 2nd Ed VCH Verlag Weinheim Federal Republic of Germany 1997 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
107. tailed 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 G rg Technical University of Munich August 1998 Table of Contents Introduction 1 0 Introduction to the manual 1 1 1 Introduction to two dimensional 2 D electrophoresis sese ese eee eel 1 1 2 Equipment choices sese sees esee 4 1 3 Laboratory technique esse eee eeees 5 Part l Sample preparation 2 0 Sample preparation general strategy 6 2 1 Methods of cell disruption 7 2 1 1 Gentle lysis methods soso soso 7 2 1 2 More vigorous lysis methods 8 2 2 Protection against proteolysis 9 2 3 Precipitation procedures 06 6005 10 2 4 Removal of contaminants that affect 2 D resultss sna kk es NE ER A 11 2 5 Composition of sample solution 12 Part Il First dimension isoelectric focusing 3 0 First dimension isoelectric focusing overview sssssssee 14 3 1 Background to isoelectric focusing IEF 14 3 2 Immobilized pH gradient selection 16 3 3 Sample application method s
108. taining buffer prepares the sample for the second dimension separation Following equilibra tion 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 elec trophoresis is 1 Sample preparation IPG strip rehydration IEF IPG strip equilibration SDS PAGE Visualization Analysis 1 2 Equipment choices Different options exist in terms of methods and equip ment for IEF and SDS PAGE Table 1 lists the instruments available from Amersham Pharmacia Biotech For detailed information on the operation of any of the instru ments described please see the respective User Manual Selecting an IEF system Amersham Pharmacia Biotech offers two different systems for the first dimension separation the Multi phor II system with associated accessories and the IPGphor Isoelectric Focusing System Multiphor II is a versatile system that can be used for several different electrophoresis techniques For 2 D electrophoresis it can be used for both first dimension IEF and second dimension SDS PAGE Strip rehydration is performed in the Immobiline DryStrip Reswelling Tray After rehydration the IPG strips are transferred to the electrophoresis unit for first dimension IEF The elec trophoresis system is comprised of the Multiphor II flatbed unit with the Immobiline DryStrip Kit Figure
109. te the sample into either or both of the Za lateral wells at either end of P the strip holder Introduce the sample below the IPG Cover Fluid Up to 7 5 pl of sample solu tion can be added to each side i e 15 nl per well or 30 pl total if both sides of both wells are used Figure 18 Note The IPG strip backing is impermeable do not apply the sample to the back of the strip Replace cover on strip holder 3 6 4 Isoelectric focusing guidelines IEF in the IPGphor system is conducted at very high voltages up to 8 000 V and very low currents typically less than 50 nA per IPG strip due to the low ionic strength within IPG strips During IEF the current FIRST DIMENSION ISOELECTRIC FOCUSING decreases while the voltage increases as proteins and other charged components migrate to their equilibrium positions A typical IEF protocol generally proceeds through a series of voltage steps that begins at a rela tively low value Voltage is gradually increased to the final desired focusing voltage which is held for up to several hours A low initial voltage minimizes sample aggregation and allows the parallel separation of samples with differing salt concentrations A gradual increase in voltage is particularly 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
110. ther single percentage or gradient the appropri ate percentage gel is selected according to the range of separation desired see Table 18 TABLE 18 RECOMMENDED ACRYLAMIDE CONCENTRATIONS FOR PROTEIN SEPARATION Acrylamide in resolving gel Separation Size Range MW x 10 Single percentage 5 36 200 1 596 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 The larger proteins fail to move significantly into the gel O Select the gel thickness Either 1 0 or 1 5 mm thick spacers can be used for all vertical formats Thinner gels stain and destain more quickly and generally give less background staining Thicker gels allow easier positioning of the IPG strip on the surface of the SDS gel and have a higher protein capacity Thicker gels are also less fragile and easier to handle e Prepare the gel solution a The total volume of solution needed depends on the gel size the gel thickness and the number of gels cast Table 19 gives volumes of gel solution required per gel for the various possible vertical gel formats TABLE 19 VOLUMES REQUIRED 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 Hoefe
111. tion is often carried out in an appropriate solubilization solution for the proteins of interest References 14 15 contain general information on tissue disruption and cell lysis 2 1 1 Gentle lysis methods Gentle lysis methods listed in Table 4 are generally employed when the sample of interest consists of easily General procedure Suspend cells in a hypoosmotic solution Freeze thaw lysis 8 14 17 Many types of cells can be lysed by subjecting them to one or more cycles of quick freezing and subsequent thawing Bacterial cells tissue culture cells Rapidly freeze cell suspension using liquid nitrogen then thaw Repeat if necessary Detergent lysis Detergents solubilize cellular membranes lysing cells and liberating their contents Tissue culture cells Suspend cells in lysis solution containing detergent 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 18 19 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 e Or separate the SDS from the sample protein by acetone precipitation See Tables 7 and 8 an
112. to four 16 cm long gels and the built in heat exchanger offers cooling capability for increased reproducibility when used with a thermostatic circulator such as MultiTemp III The standard spacer width is 2 cm giving a 14 cm wide gel If additional space for molecular weight mark ers 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 Hoefer DALT system Figure 6 is recommended The Hoefer DALT system can accommo date the entire gradient of an 18 cm IPG strip plus mole cular weight markers and up to 10 gels can be run simultaneously A built in heat exchanger and buffer circulation pump provide precise temperature control and a uniform thermal environment Twenty or more 1 or 1 5 mm thick gels can be cast simultaneously in the Hoefer DALT Multiple Gel Caster 1 3 Laboratory technique a Always wear gloves when handling IPG strips SDS polyacrylamide gels ExcelGel Buffer Strips and any equipment that these items will contact The use of gloves will reduce protein contamination that can produce spurious spots or bands in 2 D patterns 1 Clean all assemblies that will contact the gels or sample with a detergent designed for glassware and rinse well with distilled water Always use the highest quality reagents and the purest water available 2 D ELECTROPHORESIS 5 PART I Pa
113. toethanol can be used instead but higher concentrations are required and FIRST DIMENSION ISOELECTRIC FOCUSING impurities may result in artifacts 56 It has recently been reported that the non thiol reductant tributyl phosphine can be used in first dimension IEF 57 Add the reductant just prior to use gt IPG Buffer carrier ampholyte mixture can improve separations and sample solubility particularly with high sample loads IPG Buffers for each pH range are a mixture of carrier ampholytes that enhances sample solubility and produces more uniform conductivity across the pH gradient during IEF without affecting the shape of the gradient IPG Buffers are also specially formulated not to interfere with silver staining Table 9 lists the recommended final concentration of IPG Buffer for the rehydration solution The recommended IPG Buffer concentration for the IPGphor system is 0 5 but up to 2 can be added if sample solubilization remains a problem Note Concentrations at the upper end of the recom mended range may increase the time required for the voltage to reach its maximum setting during IEF which can increase the time required for complete focusing IPG Buffer can be included in the stock rehydration solution or added just prior to use IPG Buffer is included in the stock solution when multiple IPG strips of the same pH range will be used IPG Buffer is added just prior to use to single aliquots of the stock solutio
114. 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 3 5 6 Troubleshooting Table 14 lists possible problems that could be encoun tered during IEF and how to solve them TABLE 14 TROUBLESHOOTING FIRST DIMENSION IEF MULTIPHOR II AND IMMOBILINE DRYSTRIP KIT Symptom Sample cups leak Possible cause Incorrect handling and placement of sample cups Remedy Sample cups are fragile and should not be taken on and off the application bar 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 See Figure 13 Note Leaks can often be detected prior to sample application e Observe the IPG 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 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 the cup must be repositioned to eliminate the leak mportant The leak detection dye must be removed from the sample cup before loading the sample Low current This is normal for IPG gels The gels have very low conductivity Power supply cannot detect the low pA range current and shuts off IPG Buffer omitted from rehydr
115. uipment required for IEF Multiphorll 3500 V Immobiline DryStrip Reswelling Tray 3 48 hours Immobiline DryStrip Kit EPS 3501 XL power supply MultiTemp lll Thermostatic Circulator IPGphor 8000 V IPG strip holders of desired length 1 5 24 hours 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 twofold faster with the IPGphor system than with the Multiphor II system Higher voltages are not recommended for safety reasons Selecting a second dimension system The second dimension separation may be performed in a vertical or flatbed system Table 3 matches the appro priate 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 7 INTRODUCTION TABLE 3 SECOND DIMENSION ELECTROPHORESIS SYSTEM SELECTION Approx Gel IPG strip Total gel size Number thickness length oper time wxl cm ofgels mm cm h min Flatbed Multiphor Il LG sc E 0 5 all 1 45 ExcelGel 24 5 x 18 3 20 Vertical Hoefer miniVE or SE 260 8x9 2 iL ILAS 7 1 30 Hoefer SE600 14x 15 16 x 157 2 or 4 iL TES 11 13 5 00 Hoefer DALT 21 x 19 10 TES 18 7 00 15 00 Multiple shorter IPG strips fit on one ExcelGel two 11 cm strips or three 7 cm strips f 1 cm wide spacers a
116. urea non ionic detergents and IPG buffer or carrier ampholytes FIRST DIMENSION ISOELECTRIC FOCUSING 1 Apply the sample in dilute solutions 60 to 100 pg protein per 100 nl 1 Limit the voltage to 10 to 30 V cm for the initial 1 to 2 hours of focusing 1 Add Ultrodex resin to the sample 4 For micropreparative applications larger sample loads can be applied via sample cups Load the sample cup repeatedly during IEE 1 Apply the sample at both the acidic and the basic ends using two sample cup bars 3 5 3 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 A typical IEF protocol generally proceeds through a series of voltage steps that begins at a relatively low value Voltage is then gradually increased to the final desired focusing voltage which is held for up to several hours A low initial voltage minimizes sample aggregation and allows the parallel separation of samples with differing salt concentrations A gradual increase in voltage is particularly 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 conditi
117. welling of strips acidic end Dehydrated IPG strips were stored at or above room temperature for too long Incorrect volume of rehydration solution used The rehydration time is too short 3 5 2 Preparing for IEF The components of the 2 D Immobiline DryStrip Kit include a tray and electrode holder anode and cathode electrodes a 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 Clean all components of the Immobiline DryStrip Kit The Immobiline DryStrip tray DryStrip aligner elec trodes 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 e Confirm electrical connections on Multiphor II Check that the red bridging cable in the Multiphor II unit is connected e 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 Turn on MultiTemp III Thermostatic Circulator Position the Immobiline DryStrip tray Pipette approximately 10 ml of IPG 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
118. y focused spots continued Possible cause 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 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 Remedy Modify sample preparation to limit these contaminants See section 2 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 hours 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
119. y even under these conditions In these cases protease inhibitors may be used Individual protease inhibitors are active only 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 comprehensive discussions of protease inhibition see 12 28 36 40 Limitations PMSF PMSF is an irreversible inhibitor that inactivates Phenylmethylsulphonyl fluoride Most commonly used inhibitor Use at concentrations up to 1 mM serine proteases some cysteine proteases PMSF rapidly becomes inactive in aqueous solutions Prepare just prior to use PMSF may be less effective in the presence of thiol reagents such as DIT or 2 mercaptoethanol This limitation can be overcome by disrupting the sample into PMSF containing solution lacking thiol reagents Thiol reagents can be added at a later stage PMSF is very toxic AEBSF AEBSF is similar to PMSF in its inhibitory activity but is more Aminoethyl benzylsufonyl soluble and less toxic fluoride or Pefabloc SC Use at concentrations up to 4 mM AEBSF induced modifications can potentially alter the pl of a protein 1 mM EDTA or 1 mM EGTA Generally used at 1 mM metal ions required for activity These compounds inhibit metalloproteases by

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