Western Blot Hybridization(2): Buffers and Reagents
-
- Lysis buffer 1:
- 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
- 1 tablet protease inhibitor
cocktail (Roche) per 100 ml
Lysis buffer 2 (RIPA):
1% Igepal CA-630
1%
SDS
0.5% Sodium deoxycholate
Proteinase inhibitors
2x Loading buffer:
130 mM Tris-Cl, pH8.0
20%
(v/v) Glycerol
4.6% (w/v) SDS
0.02% Bromophenol blue
2% DTT
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 Transferring 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
Dilute to 1x with distilled water, with 10% methanol, before use
Blocking
buffer: 0.5 L
5% Milk
Make up in TBS and sterile filter.
Then add 0.05% Tween 20.
Keep at 4ˇăC.
Stripping buffer:
2%
Sodium Docecyl Sulfate (SDS)
100 mM 2-Mercaptoethanol
62.5 mM Trizma base, pH 6.7
10x TBS (1 L)
24.2g Trizma base
80g
Sodium Chloride
Adjust pH to 7.6
Add water to 1000 ml