Detailed Western Blotting (Immunoblotting) Protocol v1

Western Blotting, which is probably better referred to as immunoblotting, is one of the most commonly used biological methods worldwide. This technique is capable of detecting an individual protein from a complex mixture of proteins extracted from cells or tissues. The major steps in the Western Blotting workflow are 1) The separation of proteins based on size, 2) The transfer of separated protein to a suitable stable support, 3) Interaction between the target protein and appropriate primary antibodies. In many cases, a secondary antibody that interacts with the primary antibody is used, and 4) Visualization of the target protein using enhanced chemiluminescence (ECL), fluorescence, or colorimetric methods. A detailed protocol to help users get the most out of their Western Blots is presented. Goal: To help Western Blotting users learn what prevents them from having perfect Western Blots The most common result of your experiment: A good Western blot. Another possible result: No signal detected or weak signal Most likely reasons: 1. Antibody was not suitable (poor quality antibody). 2. Insufficent protein loaded on the gel for the amount of antibody used (or antibody too dluted). 3. Transfer efficiency from gel to membrane was poor. 2. ECL reagent was expired or contaminated. 5. Incorrect secondary antibody used. 6. Blocking agent concentration was too high. For more reasons read WESTERN BLOTTING TIPS AND TROUBLESHOOTING GUIDE