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Buffers for Westerns

Lysis buffer: 
0.15 M NaCl 
5 mM EDTA, pH 8 
1% Triton X100 
10 mM Tris-Cl, pH 7.4 
Just before using add: 1:1000 5 M DTT 
1:1000 100 mM PMSF in isopropanol 
1:1000 5 M e-aminocaproic acid 

2x sample buffer: 
130 mM Tris-Cl, pH8.0 
20% （v/v） Glycerol 
4.6% （w/v） SDS 
0.02% Bromophenol blue 
2% DTT 

8x Resolving gel buffer: 100 ml 
0.8 g SDS （add last） 
36.3 g Trizma base （= 3 M） 
Adjust pH to 8.8 with concentrated HCl 

4x Stacking gel buffer: 100 ml 
0.4 g SDS （add last） 
6.05 g Trizma base （= 0.5 M） 
Adjust pH to 6.8 

10x Running buffer: 1 L 
30.3 g Trizma base （= 0.25 M） 
144 g Glycine （= 1.92 M） 
10 g SDS （= 1%）--add last 
Do not adjust the pH!! 

10x Blotting buffer: 1 L 
30.3 g Trizma base （= 0.25 M） 
144 g Glycine （= 1.92 M） 
pH should be 8.3; do not adjust 

To make 2 L of 1x Blotting buffer: 
400 ml Methanol 
200 ml 10x Blotting buffer 
1400 ml water 

Blocking buffer: 0.5 L 
3% Bovine serum albumin （Fraction V） 
Make up in PBS and sterile filter. 
Then add 0.05% Tween 20. 
Keep at 4°C to prevent bacterial contamination. 

Stripping buffer: 0.5 L （sterile filter solution and keep at 4°C） 
0.2 M Glycine, pH 2.5 
0.05% Tween 20