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P.1

Protocol P.1

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 ml of this mix with 250 ml b-Mercaptoethanol

 

Coomasie Protein Stain Solution

0.04% Coomasie Blue 0.17 g Coomasie Blue (Biorad #161-0406)

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 AgNO3 (Sigma #S0139)

100 ml Q

store at room temperature in a light proof bottle

Silver Stain Fixer

50% MeOH 250 ml MeOH

12% acetic acid 60 ml acetic acid

0.0185% formaldehyde 250 ml 37% formaldehyde

Q to 500 ml

 

 

 

Silver Stain Developer

6% Na2CO3 12 g Na2CO3

0.004% sodium thiosulfate 4 ml 0.02% sodium thiosulfate

0.0185 % formaldehyde 100 ml 37% formaldehyde

 

 

 

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 5 ml 5 ml 5 ml

ACRYLAMIDE (29:1) 5 ml 6.7 ml 8 ml

Q 10 ml 8.3 ml 7 ml

Add 300 ml 10% APS and 30 ml TEMED. Pour gels allowing 1.5 cm for the stacking gel. Overlay the resolving gel with 0.2% SDS (approx. 200 ml for 0.75 mm gels). The gels will polymerize in approximately 5 minutes.

• Mix a 3% stacking gel:

2.5 ml 4X Stacking Gel Buffer

6.5 ml Q

1.0 ml Acrylamide (29:1)

• Add 200 ml 10% APS and 20 ml TEMED. Pour out the 0.2% SDS overlay and wash the top of the gel with approximately 1ml of the stacking gel mix. Pour the stacking gel, insert comb. The stacking gel will polymerized in approximately 10 minutes.

• 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 ml 37% formaldehyde for 20 minutes.

• 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.