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Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics
Modified Protocol for Western Blotting (Page 1, 2, 3)--Western Blotting Center
Buffers for Western Blotting
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
(This protocol modified from the one of Howell Lab,