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Protein Gel Preparation and Staining
This is a protocol using the glycine system described by Laemmli, U.K. (1970) Nature 227:680-685. For polypeptides smaller than 30 kDa, the tricine system described by Schagger, H. & Von Jagon, G. (1987) Analytical Biochemistry 166:368-379) is preferable. Solutions 30% Acrylamide 29 g acrylamide 1 g Bis acrylamide up to 100 ml with Q store at room temperature in a light proof bottle 4X Resolving Gel Buffer 1.5 M Tris pH 8.8 18.17 g Tris Base pH to 8.8 with HCl 0.4% SDS 0.4 g SDS up to 100 ml with Q 4X Stacking Gel Buffer 0.5 M Tris pH 6.8 6.06 g Tris Base pH to 6.8 with HCl 0.4% SDS 0.4 g SDS up to 100 ml with Q 1X Glycine Running Buffer 250 mM Tris Base 1.65 g Tris Base 250 mM glycine 9.35 g glycine 0.1% SDS 2.5 ml 20% SDS up to 500 ml with Q 5X Loading Dye 0.5 M Tris 6.8 5 ml 1M Tris pH 6.8 50% glycerol 5 ml glycerol 0.5% bromophenyl blue 0.05 g bromophenyl blue 10% SDS 1 g SDS To use: Mix 750 Coomasie Protein Stain Solution 0.04% Coomasie Blue 0.17 g Coomasie Blue 25% isopropanol 60 ml isopropanol 10% acetic acid 25 ml acetic acid up to 250 ml with Q this can be used for several months, store air tight Coomasie Destain Solution 10% acetic acid 100 ml acetic acid 10% methanol 100 ml methanol up to 1 liter with Q 20% Silver Nitrate 20 g AgNO 100 ml Q Silver Stain Fixer 50% MeOH 250 ml MeOH 12% acetic acid 60 ml acetic acid 0.0185% formaldehyde 250 Q to 500 ml Silver Stain Developer 6% Na 0.004% sodium thiosulfate 4 ml 0.02% sodium thiosulfate 0.0185 % formaldehyde 100 Procedure pour minigels • Mix buffer, acrylamide and Q as indicated below for 2 minigels (0.75 or 1.5 mm) and double the recipe for 1 large gel. GEL PERCENTAGE 7.5% 10% 12% 4X RESOLVING BUFFER ACRYLAMIDE (29:1) Q • • Mix a 3% stacking gel: 6.5 ml Q 1.0 ml Acrylamide (29:1) • Add 200 • Boil the samples in SDS Loading dye for 2 minutes and place immediately on ice. If all of the lanes are not needed, prepare dye samples for the empty lanes to prevent distortions. Run the minigels at 75-200 Volts, constant voltage. coomasie dye staining • Place gel(s) in coomasie dye mixture and stain with shaking for 1 hr to overnight. • Remove gel(s) and place in destain along with several kimwipes. • Rinse in water and dry between two sheets of celophane or on gel drier mounted on Whatmann 3mm paper. silver staining • Fix gel overnight in 500 ml Fix Solution. • Wash the gels 3 times in 50% MeOH (330 ml each wash). • Pretreat in 500 ml freshly made sodium thiosulfate (0.02%) for 1 minute. Save some of this for later (0.2 g/liter Q). • Rinse in Q, 3 times at 20 seconds each (330 ml per wash). • Stain with 200 ml fresh 0.2% Silver Nitrate containing 150 • Rinse in Q, 2 times at 30 seconds each (500 ml per wash). • Develop with 200 ml fresh Developing Solution until the bands appear. • Rinse in Q and stop the reaction in 50% MeOH/12% acetic acid. • Rinse in Q and dry between two sheets of celophane or on a gel drier mounted on Whatmann 3mm paper.
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