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.
Why does my IP reaction still appear as a slurry and not a pellet after spinning?
The IP reaction should only be spun at a speed of 200 x g to 500 x g (maximum 2400 rpm in a microfuge). Spinning at higher speeds will results in rupture of the protein A/G beads and a pellet will not form.
Do I have to use protease inhibitors in the lysis buffer used in the wash steps of the IP reaction?
Inhibitors are not necessary for the wash the steps.
How do I know how much antibody to use in the immunoprecipitation reaction?
For best results, the optimal amounts of antibody should be empirically determined. But a general rule is to add 2 to 10 micrograms of antibody per 500 micrograms of lysate. If you are using neat antisera, or an IgG fraction (such as protein-A purified antibody), greater amounts of antibody are likely to be required.