application protocols

Western Blot Protocol

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.

Reagents mentioned in this Western Blot protocol

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

*Note:Block Ace is recommended over 5% non-fat dried milk dissolved in PBS for cleaner Western blots. For the detection of phosphoproteins, NEVER block membranes with milk; use 5% BSA instead. Milk contains casein (itself a phosphoprotein) which will bind phospho-specific antibodies resulting in high background.
Washing Buffer
Blocking buffer + 0.1% Tween®-20
Ponceau S
Ponceau S (Sigma P3504), 0.1 g
Acetic Acid, 5 ml
Distilled Water, 95 ml

*Note: Ponceau S is light sensitive.
PBS
Sodium Chloride, 8.0g  Potassium Chloride, 0.2g
Disodium Potassium Phosphate, 1.15g
Potassium Dihydrogen Phosphate, 0.2g
Distilled Water, 1 liter
PBST
Sodium Chloride, 8.0g
Potassium Chloride, 0.2g
Disodium Potassium Phosphate, 1.15g
Potassium Dihydrogen Phosphate, 0.2g
Tween-20®, 1.0ml
Distilled Water, 1 liter

Method

  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 minute, then completely destain the blot by washing with distilled water.
  3. Place blot into blocking solution for 2 hours at room temperature, 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 hours at room temperature, or overnight at 4°C.
  5. Wash the blot extensively in wash buffer (3 x 10 minutes) with gentle agitation.
  6. Add appropriate enzyme-conjugated secondary antibody diluted in wash buffer and incubate for 1 hour at room temperature with gentle agitation.
  7. Wash the membrane with gentle agitation as follows: 4 x 5 minutes in wash buffer; 3 x 5 minutes in PBST and 2 x 5 minutes 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 non-specific binding of the secondary reagent to the target tissue. Please contact Bio-Rad’s Technical Services Department to learn about recoommended 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.