Sample Protocol

Separating Gel Buffer or Lower Tris

1. 1.5 M Tris-HCl pH 8.8
   
2. 0.4% SDS
   

Stacking Gel Buffer or Upper Tris

1. 0.5 M Tris-Cl, pH 6.8
   
2. 0.4% SDS
   

Acrylamide Stock

1. 30% (w/v) acrylamide
   
2. 0.8% (w/v) bis
   

Sample Buffer- 2X

1. 50% Stacking gel buffer
   
2. 20% glycerol (v/v)
   
3. 4% (w/v) DTT or 10% (v/v) ß-mercaptethanol
   
4. 0.02% Bromophenol blue
   

Running Buffer

1. For 1x - 0.025 M Tris-glycine, pH 8.9
   0.1% SDS
   
2. For 5x - 0.125 M Tris-glycine, pH 8.9
   0.5% SDS

Coomassie Stain

To make Coomassie staining solution you will need:

1. 400mL of water
   
2. 500mL of methanol
   
3. 1g of Coomassie Blue
   
4. 100mL of acetic acid
   

1. Mix together for one hour

2. Add 100mL of acetic acid

3. Filter solution into another container

4. Stain gel(s) for 1 hour

Destaining Protocol

To make destaining solution you will need:

1. 600mL of water
   
2. 300mL of methanol
   
3. 100mL of acetic acid
   
4. Kim wipes, or some other absorbent material
   

Destain for 1 hour, occasionally removing/ replacing kimwipes as they become saturated w/ Coomassie.

In-Gel Digestion Protocol

Short and long methods of in-gel digestion

TBS (Tris Buffered Saline)

1. 10 mM Tris pH 7.4
   
2. 150 mM NaCl
   

TBS-Triton

1. 10 mM Tris, pH 7.4
   
2. 150 mM NaCl
   
3. 0.05% Triton X-100
   

Blocking Buffer

1. 10 mM Tris, pH 7.4
   
2. 150 mM NaCl
   
3. <3% to 4% BSA (Bovine Serum Albumin) or 5 &endash; 8% milk./li>
   

Ponceau S Stain

1. Make 1: 10 dilution in water from concentrate
   
2. Ponceau S 2%
   
3. Trichloroacetic acid 30%
   
4. Sulfosalicylic acid 30%