Immunoprecipitation (IP)-Usage of IP antibody & Cell Lysate

Questions & Solutions

A. When I perform a western on immunoprecipitated samples, do I need to use more or less primary antibody when probing the western blot?

When probing immunoprecipitated samples on a western blot, the concentration of primary antibody can be increased resulting in an increase in sensitivity. However, for best results, the optimal dilution of antibody should be empirically determined.

B. How much antibody and how much sample should be used?

Usually, we suggest using 10-500 μg cell lysate per sample plus the recommended amount of antibody. These amounts will be chosen depending on the abundance of the target protein and the affinity of the antibody for the protein, typically in a pre-experiment where a fixed amount of protein is precipitated by increasing amounts of antibody. 
You can check the IP antibody datasheet for recommended antibody concentration. As a guideline use:
1 – 5 μl polyclonal antiserum
1 μg affinity-purified polyclonal antibody
0.2 to 1 μl ascites fluid (monoclonal antibody) 
20 to 100 μl culture supernatant (monoclonal antibody)

C. Too many cells/too much protein in lysate leading to a lot of non-specific proteins in eluate

Reduce the number of cells/lysate used . Generally using 10-500 µg cell lysate is enough . Besides, you can also choose goat serum or normal goat IgG for blocking.

D. Too much antibody used leading to non-specific binding

Check the recommended amount of antibody suggested. Try using decrease antibody by gradient for the best target band .