Buffers / Reagents Preparation for Western Blot

In 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 solution are 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, TBS buffer, blocking buffer, and TBST buffer (washing buffer) are also needed to be prepared. The table below is a recipe especially about buffer or reagent needed in western blot, or we can name this table after "western blot buffer recipe".

Recipes for Western Blot buffers

Buffer category Buffer name Recipe
Basic buffers 10X TBS buffer (pH 7.6) For 1.0 L: 24.2 g Tris-base
87.8 g NaCl
Adjust the pH to 7.6, and add ddH2O to 1 L.
10X PBS buffer (pH 7.4) For 1.0 L: 80.0 g NaCl
2.0 g KCl
14.4 g Na2HPO4
2.4 g KH2PO4
Adjust the pH to 7.4, and add ddH2O to 1 L.
Cell lysis buffers NP-40 lysis buffer 150 mM NaCl
50 mM Tris-HCl, pH 8.0
1.0% NP-40 or Triton X-100

Add fresh protease inhibitors before use.
RIPA (Radioimmunoprecipitation Assay) lysis buffer 140 mM NaCl
10 mM Tris-HCl, pH 8.0
1 mM EDTA
0.5 mM EGTA
1% Triton X-100 or NP40
0.1% sodium deoxycholate
0.1% SDS

Add fresh protease inhibitors before use.
Tris–Triton lysis buffer 10 mM Tris, pH 7.4
100 mM NaCl
1 mM EDTA
1 mM EGTA
1% Triton X-100
10% glycerol
0.1% SDS

Add 0.5% deoxycholate, 1:200 protease inhibitor cocktail, and 1% phosphatase inhibitor before use.
SDS-PAGE buffers & solutions 2X Laemmli loading buffer (reducing) 120 mM Tris-HCl, pH 6.8
4% SDS
20% glycerol
10% 2-mercaptoethanol

Add bromophenol blue to a final concentration of 0.02% (w/v) before use.
SDS-PAGE 10X running buffer For 1.0 L: 30.0 g Tris base
144.0 g glycine
10.0 g SDS
Do not adjust the pH!
Add ddH2O to 1 L.
Coomassie Brilliant Blue solution For 1.0 L: 1.0 g Coomassie brilliant blue
400 mL methanol
100 mL acetic acid
Add ddH2O to 1 L.
Coomassie destaining solution For 1.0 L: 200 mL methanol
100 mL acetic acid
Add ddH2O to 1 L.
Transfer buffer & solutions Western Blot 1X transfer buffer (wet) For 1.0 L: 3.0 g Tris-base
14.4 g glycine
200 mL methanol
Add ddH2O to 1 L.
Western Blot 1X transfer buffer (semi-dry) For 1.0 L: 5.76 g Tris-base
2.95 g glycine
200 mL methanol
Add ddH2O to 1 L.
Ponceau S staining solution For 1.0 L: 0.5 g Ponceau S
25 mL acetic acid
Add ddH2O to 1 L.
Western Blot washing buffer 1X PBST (Phosphate-buffered saline, 0.1% Tween 20) For 1.0 L: 100 mL 10X PBS
899 mL ddH2O
1 mL Tween 20
1X TBST (Tris-buffered saline, 0.1% Tween 20) For 1.0 L: 100 mL 10X TBS
899 mL ddH2O
1 mL Tween 20
Blocking buffer Western Blot blocking buffer 5% (or 3%) nonfat dried milk or BSA in 1X TBST or 1X PBST.