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