Multiple Mini-Format 2-D Electrophoresis using SE 600 Standard
Contents
1. apy lication note 2 D Electrophoresis Multiple Mini Format 2 D Electrophoresis SE 600 Standard Vertical Unit Keywords 2 D Electrophoresis 7 cm IPG Drystrip Two dimensional 2 D electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures With the recent improvements to and uses for this technique the need for higher throughput has increased This application note describes a method for performing the second dimension separation for two to eight mini format 2 D gels simultaneously in a single vertical electrophoresis unit The procedure yields high quality separations using 7 cm Immobiline DryStrip IPG gel strips and the SE 600 Dual Vertical Gel unit This arrangement simplifies and improves differential compar isons Using a third glass plate and additional spacers for the SE 600 a club sandwich assembly can be formed which produces two identical gels in a single gel space Thus eight second dimension separations can be performed on one unit two IPG strips per gel two gels per sandwich and two sandwiches per unit At approximately three hours per run 16 mini format 2 D separations can be completed in a normal workday on a single SE 600 system Protocol 9 Casting gels 1 1 Assemble the SE 600 vertical slab gel unit in the dual gel casting stand using 18 x 8 cm glass plates 80 6186 59 For running eight 2 D mini gels simultaneously use the glass plate club sandwich divi2. 1 Amersham e Biosciences 2 D Electrophoresis 2 Equilibrate the IPG strip IPG strips are run and equilibrated for application to the second dimension as described in the 2 D Principles handbook Soak strips for 15 min in equilibration solu tion solution A containing 1 w v DTT followed by 15 min more in equilibration solution containing 2 5 w v iodoacetamide Two 7 cm long IPG strips can be equili brated simultaneously in a 15 ml conical centrifuge tube using 6 ml of equilibration solution Apply the equilibrated IPG strip 3 1 Place the IPG strip Dip the IPG strip in the SDS electrophoresis buffer solu tion B to lubricate it Position the first IPG strip between the plates on the surface of the second dimension gel with the plastic backing against one of the glass plates Be sure to place the strip as far as possible to one side of the gel With a thin plastic ruler 80 6223 83 gently push the IPG strip down so its entire lower edge is in contact with the top surface of the slab gel Ensure that no air bubbles are trapped between the IPG strip and the slab gel surface or between the gel backing and the glass plate Repeat this procedure placing the second strip next to the first and leaving a space of approximately 1 cm between the strips 3 2 Optional Apply molecular weight marker proteins The markers are applied in 0 5 agarose M or NA to a paper IEF sample application piece in a vo
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4. at 2 D separations can be run in a normal workday on a single SE 600 system For more detailed information and troubleshooting of 2 D electrophoresis see the 2 D Principles handbook Fig 2 Multiple Mini Format 2 D Gel E coli extract 24 ug pH 3 10 NL first dimension 1 mm thick 12 5 acrylamide second dimension Table 1 Volumes of Acrylamide Required for Mini Format 2 D Gels Total Volume ml Number of 16 x 8 cm Gels 2 4 1 5 mm thick spacers 40 80 1 mm thick spacers 30 60 Solutions required A SDS equilibration buffer 50 mM Tris Cl pH 8 8 6M urea 30 glycerol 2 SDS bromophenol blue 200 ml Final concentration Amount 1 5 M Tris Cl pH 8 8 50 mM 6 7 ml solution D Urea FW 60 06 6M 72 02 g Glycerol 87 v v 30 v v 69 ml SDS FW 288 38 2 wiv 4g Bromophenol blue trace a few grains Double distilled H20 to 200 ml 1This is a stock solution Prior to use DTT or iodoacetamide is added See section 2 Store in 40 ml aliquots at 20 C B SDS electrophoresis buffer 25 mM Tris 192 mM glycine 0 1 SDS 5 litres Final concentration Amount Tris base FW 121 1 25 mM 15 1 g Glycine FW 75 07 192 mM 72 1 SDS FW 288 38 0 1 w v 5 0g Double distilled H20 to 5 000 ml 1 Because you do not need to titrate this buffer to a specific pH it can be made up directly in large reagent bottles marked at 5 litres 20 litres can be made up at a time Store at r
5. d 0 5 litre for the cathode Q Electrophoresis conditions Electrophoresis is performed at constant current in two steps Start at 15 mA gel for an initial migration and stacking period of 15 min then increase to 30 mA gel for a period of approximately 3 h Currents up to 50 higher may be used during the second separation phase if only two gels per unit are being run no club sandwich dividers and the unit is being cooled with a thermostatic circulator Stop the electrophoresis when the dye front is approxi mately 1 mm from the bottom of the gel Cooling is optional however temperature control improves gel to gel reproducibility especially if the ambi ent temperature of the laboratory fluctuates significantly Do not cool SDS containing gels below 15 C After electrophoresis remove gels from their 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 2 D Electrophoresis Conclusions This multiple mini format method produces high quality 2 D separations Fig 2 and allows for two to eight mini format 2 D gels to be run on a single SE 600 unit Differential comparisons are improved by this method by minimizing gel to gel variations In addition this method allows for increased throughput with minimal equipment At approximately three hours per second dimension run 16 mini form
6. ders 80 6186 78 The divider plates allow two gels to be cast in a single sandwich Be sure to use the 8 cm x 1 cm x 1 mm 80 6443 09 or 8 cm x 1 cm x 1 5 mm 80 6443 28 spacers 1 2 Cast the gels following the instructions provided in the SE 600 Series User Manual 80 6353 79 or 2 D Elec trophoresis Using Immobilized pH Gradients Principles amp Methods 80 6429 60 2 D Principles handbook Appropriate volumes of acrylamide are listed in Table 1 80 6445 94 Rev B 9 99 pl Fig 1 SE 600 and clamp assembly Products used SE 600 Dual Cooled Vertical Unit 80 6171 58 Glass plates 18 x 8 cm 2 pk 80 6186 59 Glass plate club sandwich divider notched 18 x 8 cm 80 6186 78 Clamp assembly 8 cm 2 pk 80 6187 35 Spacer 1 0 mm 1 cm wide 8 cm long 2 pk 80 6443 09 Spacer 1 5 mm 1 cm wide 8 cm long 2 pk 80 6443 28 EPS 601 Power Supply 18 1130 02 Plastic ruler 80 6223 83 MW markers 2 512 16 949 range 80 1129 83 MW markers 14 400 94 000 range 17 0446 01 IEF Sample Application Pieces 200 pk 80 1129 46 PlusOne Reagents Urea 500 g 17 1319 01 Tris 500 g 17 1321 01 SDS 100 g 17 1313 01 Glycerol 87 1 litre 17 1325 01 Bromophenol blue 17 1329 01 Glycine 500 g 17 1323 01 Agarose M 10 g 17 0422 01 Agarose NA 10 g 17 0554 01 Dithiothreitol DTT 1 g 17 1318 01 Acrylamide IEF 40 solution 1 000 mi 17 1301 01 N N methylenebisacrylamide 25 g 17 1304 01 Ammonium persulphate 25 g 17 1311 0
7. lume of 15 to 20 ul For less volume cut the sample application piece proportionally Add 0 5 agarose to the appropriate volume of marker solution and heat at 100 C for approxi mately 5 min 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 in the space between the two IPG strips The markers should contain 200 to 1 OOO ng of each component for Coomassie staining and about 10 to 50 ng of each component for silver staining 3 3 Seal the IPG strip in place Embedding the IPG strips in agarose prevents them from moving or floating in the electrophoresis buffer Prepare agarose sealing solution solution C Melt each aliquot as needed in a 100 C heat block each gel will require 0 5 to 1 mi It takes approximately 10 min to fully melt the agarose Tip An ideal time to prepare and melt the agarose is during the IPG strip equilibration step 2 Allow the agarose to cool to 40 to 50 C and then slowly pipette onto the top of the gel the amount required to seal the IPG strip in place Pipetting slowly avoids introducing bubbles Allow a minimum of 1 min for the agarose to cool and solidify 3 4 Finish assembling the electrophoresis unit see the SE 600 Series User Manual 80 6353 79 and add SDS electrophoresis buffer solution B This method will require a total of 5 litres of buffer 4 5 litres for the anode an
8. oom temperature continued on page 4 p3 2 D Electrophoresis Solutions required continued C Agarose sealing solution Final concentration Amount SDS electrophoresis buffer 100 ml solution B Agarose NA or M 0 5 0 5g Bromophenol blue trace 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 D 1 5 M Tris Cl pH 8 8 Final concentration Amount Tris base FW 121 1 1 5 M 181 5 g Double distilled H20 750 ml HCI FW 36 46 adjust to pH 8 8 Double distilled H20 to 1 000 ml Filter solution through a 0 45 um filter Store at 4 C For more information visit the Amersham Phamacia Biotech Website www amershambiosciences com Immobiline and PlusOne are trademarks of Amersham Biosciences Limited or its subsidiaries Amersham and Amersham Biosciences is a trademark of Amersham plc All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request 1999 Amersham Biosciences UK Limited All rights reserved Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE 751 84 Uppsal
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