When we do western blot, except lysis buffer which is needed in sample preparation, other reagents also have to be prepared for western blot. Loading buffer, running buffer, coomassie brilliant blue staining solution, and coomassie destaining solutionare needed to be prepared for SDS-PAGE, while western blot transfer buffer (recipe here is for wet transfer) preparation is required for protein transfer. Sometimes, ponceau red staining is an alternative to check whether the protein transfer is successful, so a recipe of ponceau red staining solution is necessary. Besides, we also need TBS buffer, blocking buffer, and TBST buffer (washing buffer). there are some recipes especially about buffer or reagent needed in western blot.
(1) 2×Loading Buffer
0.125 M Tris-HCl (pH 6.8)
4% SDS
20% glycerol
0.004% Bromophenol blue
10% 2-mercaptoethanol
(2) SDS-PAGE Running Buffer
Tris-base 3.0 g
Glycine 14.4 g
10% SDS 10 mL
Add ddH2O to 1 L,
do not adjust the pH!
(3) Coomassie Brilliant Blue Solution
Coomassie brilliant blue 1 g
Methanol 400 ml
Acetic acid 100 ml
ddH2O 500 ml
Total 1 L
(4) Coomassie Destaining Solution
Methanol 200 ml
Acetic acid 100 ml
ddH2O 700 ml
Total 1 L
(5) Western Blot Transfer Buffer
Tris-base 3.0 g
Glycine 14.4 g
Methanol 200 mL
Add ddH2O to 1 L
(6) Ponceau Red Staining Solution
Ponceau S 0.5 g
Acetic acid 25 ml
ddH2O 475 ml
Total 1 L
(7) TBS Buffer(pH 7.4)
Tris-base 2.42 g
NaCl 8.78 g
Add ddH2O to 1 L,
adjust the pH to 7.4
(8) Western Blot Blocking Buffer
5% milk or BSA (bovine serum
albumin)
Add to TBS buffer.
Mix well and filer.
(9) Western Blot Washing Buffer
(TBST Buffer)
0.05% Tween-20 in TBS buffer
Add 0.25 mL Tween-20 to 500 mL
TBS buffer.
Western Blot 