Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4232 Protocol Multiple SDS-PAGE on Vertical Electrophoresis Units Angelika Görg, Oliver Drews, and Walter Weiss This protocol was adapted from "Separation of Proteins Using Two-dimensional Gel Electrophoresis," contributed by Angelika Görg, Oliver Drews, and Walter Weiss, Chapter 16, in Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004. INTRODUCTION SDS-polyacrylamide gel electrophoresis (SDS-PAGE) represents the second-dimension separation of two-dimensional (2D)-PAGE. Large-scale proteome analysis usually requires simultaneous electrophoresis of batches of second-dimension SDS-PAGE gels to maximize the reproducibility of 2D electrophoresis protein profiles. This requirement is most easily met using multiple, vertical second-dimension SDS-PAGE systems. This protocol presents a method for preparing and running multiple vertical SDS gels. MATERIALS Reagents 2D-PAGE agarose solution Bromophenol blue [0.25% (w/v) in IPG Tris buffer] Dithiothreitol (DTT) Focused IPG strips (see Isoelectric Focusing in Immobilized ph Gradient Strips Using the IPGphor Unit: Sample In-Gel Rehydration and Isoelectric Focusing in Immobilized ph Gradient Strips Using the IPGphor Unit: Sample Cup Loading) Iodoacetamide IPG equilibration buffer Molecular weight markers for SDS gels Ready-made DALT Gel 12.5 (optional) (Amersham Biosciences) Equipment Cassette rack (e.g., Amersham Biosciences) A cassette rack is recommended for storing gel cassettes in an upright position to facilitate the application of the IPG gel strips.
DALT gel cassettes (Amersham Biosciences) Ettan-DALT II multiple vertical electrophoresis unit (Amersham Biosciences) Glass test tubes for equilibration (200-250 mm long, 20 mm I.D.) (e.g., Amersham Biosciences) Laboratory shaker Parafilm Power supply Staining trays made from glass (e.g., Pyrex) or stainless steel (e.g., Amersham Biosciences) Thin spatula or ruler Ready-made DALT SDS gels (Amersham Biosciences) METHOD Equilibration of IPG Gel Strips 1. Dissolve 100 mg of DTT in 10 ml of IPG equilibration buffer to make Equilibration Buffer A. Make 10 ml per sample. Place the focused IPG gel strips into individual test tubes. Add 10 ml of Equilibration Buffer A and 50 µl of bromophenol blue solution to each tube. Seal the tubes with Parafilm, rock them for 15 minutes on a shaker, and pour off the equilibration buffer. SDS, urea, and glycerol improve protein transfer into the SDS gel. Dithiothreitol (DTT) reduces disulfide bonds. 2. Dissolve 0.4 g of iodoacetamide in 10 ml of equilibration buffer to make Equilibration Buffer B. Make 10 ml per sample. Add this buffer and 50 µl of bromophenol blue solution to each tube, and equilibrate for another 15 minutes with gentle agitation. Iodoacetamide is included instead of DTT to remove excess DTT and to alkylate SH groups, which is crucial if sample spots are to be analyzed by mass spectrometry. 3. Pour off Equilibration Buffer B. SDS-PAGE 4. Add 1875 ml of electrode buffer stock solution and 5625 ml of deionized H 2 O to the lower electrophoresis buffer tank of the Ettan-DALT II unit. Mix, and turn on cooling (20ºC). 5. Support the DALT gel cassettes (containing the SDS gels) in a vertical position on the cassette rack to facilitate the application of the IPG gel strips. 6. Briefly rinse each equilibrated IPG strip with electrode buffer (diluted 1:1 with H 2 O), and place an IPG strip on top of the DALT gel cassette. Use a thin spatula or a ruler to push against the plastic backing of the IPG strip, and slide it into the gap between the two glass plates. Add 2 ml of hot (75ºC) agarose solution, and continue to slide the strip down onto the surface of the SDS gel until good contact is achieved. Avoid trapping air bubbles between the IPG strip and the SDS gel surface.
Repeat with each IPG strip. EXPERIMENTAL TIP: For coelectrophoresis of molecular weight marker proteins, soak a filter paper pad (2 x 4 mm 2 ) with 5 µl of SDS marker proteins dissolved in electrophoresis buffer. Allow it to dry, and apply it to the left or right of the IPG strip. 7. Allow the agarose to solidify for at least 5 minutes before placing the slab gel into the electrophoresis apparatus. 8. Wet the outside of the gel cassettes by dipping them into electrode buffer for a few seconds to make them fit more easily into the electrophoresis unit. Insert them in the electrophoresis apparatus. If necessary, push blank cassette inserts into any unoccupied slots. Seat the upper buffer chamber over the gels, and fill it with 2.5 liters of 0.5X electrode buffer. 9. Place the safety lid on the electrophoresis unit, and start SDS-PAGE with 5 ma per SDS gel (100 V maximum setting) for approximately 2 hours. Continue with 15 ma per SDS gel (200 V maximum setting) for approximately 16 hours. 10. Terminate the run when the bromophenol blue tracking dye has migrated off the lower end of the gel. 11. After electrophoresis, carefully open the cassettes with a plastic spatula. Use the spatula to separate the agarose overlay from the polyacrylamide gel. Carefully peel the gel from the glass plate, lifting the gel by its lower edge, and place it in a box of fixing or staining solution. Caution Bromophenol blue Bromophenol blue may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses and use in a chemical fume hood. Caution Dithiothreitol (DTT) Dithiothreitol (DTT) is a strong reducing agent that emits a foul odor. It may be harmful by inhalation, ingestion, or skin absorption. When working with the solid form or highly concentrated stocks, wear appropriate gloves and safety glasses and use in a chemical fume hood.
Caution Iodoacetamide Iodoacetamide C 2 H 4 INO can alkylate amino groups in proteins and can therefore cause problems if the antigen is being purified for amino acid sequencing. It is toxic and harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses and use only in a chemical fume hood. Do not breathe the dust. 2D PAGE agarose solution Agarose Bromophenol blue Suspend 1.0 g of agarose in 90 ml of electrode buffer and 100 ml of deionized H 2 O and melt it in a boiling water bath or in a microwave oven. Then add 10 ml of bromophenol blue solution (50 mg of bromophenol blue dissolved in 10 ml of electrode buffer). 96 mm Tris base 500 mm glycine 0.4% (w/v) SDS To make 5 liters of electrode buffer stock solution, dissolve 58.0 g of Tris base, 299.6 g of glycine and 19.9 g of SDS in deionized H 2 O. Adjust the volume to 5.0 liters.
IPG Equilibration buffer 6 M urea 30% (w/v) glycerol 2% (w/v) SDS in 0.05 M IPG Tris-HCl buffer (ph 8.8) To make 500 ml, combine 180 g of urea, 150 g of glycerol, 10 g of SDS, and 16.7 ml of IPG Tris-HCl buffer. Dissolve in deionized H 2 O and adjust the volume to 500 ml. This buffer can be stored at room temperature for up to 2 weeks. IPG Tris-HCl buffer (ph 8.8) 1.5 M Tris base 0.4% (w/v) SDS 4 N HCl sodium azide To make 25 ml, dissolve 4.55 g of Tris base and 0.1 g of SDS in ~20 ml of deionized H 2 O. Adjust the ph of the solution with 4 NHCl and adjust the volume to 25 ml with deionized H 2 O. Add 2.5 mg ofsodium azide and filter. The buffer can be stored at 4 C for up to 2 weeks. Copyright 2006 by Cold Spring Harbor Laboratory Press. Online ISSN: 1559-6095 Terms of Service All rights reserved. Anyone using the procedures outlined in these protocols does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in these protocols and has no liability in connection with their use. All materials used in these protocols, but not limited to those highlighted with the Warning icon, may be considered hazardous and should be used with caution. For a full listing of cautions, click here. All rights reserved. No part of these pages, either text or images, may be used for any reason other than personal use. Reproduction, modification, storage in a retrieval system or retransmission, in any form or by any means-electronic, mechanical, or otherwise-for reasons other than personal use is strictly prohibited without prior written permission. CiteULike Connotea Del.icio.us Digg Reddit Technorati What's this?