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Ch 13.HPLC and TLC

Ch 14.Buffers formats in Lab.

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Modified Protocol for Western Blotting (Page 123)--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, UCSD, USA)

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