This western blot protocol provides a general procedure for use with the majority of Bio-Rad reagents. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with product and batch specific information provided with each vial.

Please note that a certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques - these are guidelines only and may need to be adjusted for particular applications.



Blocking Buffer
Block Ace BUF029 dissolved in water, or 5% non-fat dried milk dissolved in PBS.

*Note: For cleaner western blots, Block Ace is recommended over 5% non-fat dried milk dissolved in PBS. β€‹For the detection of phosphorylated protein, use the recommended blocking solution as stated on the product datasheet. However, we advise using our protocol for detection of phosphorylated proteins by western blot.
Washing buffer
Blocking buffer + 0.1% Tween 20
Ponceau S
Acetic acid, 5 ml
Distilled water, 95 ml
Ponceau S (Sigma P3504), 0.1 g

*Note: Ponceau S is light sensitive.
Disodium potassium phosphate, 1.15g
Distilled water, 1 L
Potassium chloride, 0.2 g
Potassium dihydrogen phosphate, 0.2g
Sodium chloride, 8.0 g  
Disodium potassium phosphate, 1.15 g
Distilled water, 1 L
Potassium chloride, 0.2 g
Potassium dihydrogen phosphate, 0.2g
Sodium chloride, 8.0 g
Tween 20, 1.0 ml


  1. Following SDS-PAGE, transfer proteins onto blotting membrane according to the manufacturer’s instructions.
  2. Check protein transfer by staining the blot with Ponceau S for 1 min, then completely destain the blot by washing with distilled water.
  3. Place blot into blocking solution for 2 hr at RT, or overnight at 4°C.
  4. Rinse the blot briefly with wash buffer and then add primary antibody diluted in the wash buffer (a concentration of 1-10 µg/ml is generally acceptable, but check datasheets for precise recommendations). Incubate for 2 hr at RT, or overnight at 4°C.
  5. Wash the blot extensively in wash buffer (3 x 10 min) with gentle agitation.
  6. Add appropriate enzyme-conjugated secondary antibody diluted in wash buffer and incubate for 1 hr at RT with gentle agitation.
  7. Wash the membrane with gentle agitation as follows: 4x 5 min in wash buffer; 3x 5 min in PBST and 2x 5 min in PBS.
  8. Add appropriate enzyme substrate solution and incubate as recommended by the manufacturer to visualize protein bands.

Appropriate controls should always be carried out. It may be useful to include a sample in which no primary antibody is used at all, in order to determine any nonspecific binding of the secondary reagent to the target tissue. Please contact Bio-Rad’s Technical Services Department to learn about recommended secondary reagents for specific applications.

Western Blot Video: SDS-PAGE Separation of Proteins

This video demonstrates SDS-PAGE separation of proteins using the Bio-Rad Comparative Proteomics Kit II: Western Blot Module.

Assembly of the blotting sandwich and electroblotting are shown along with the steps for protein detection using a colorimetric assay.