Help for immunoprecipitation - (May/04/2005 )
It is frustrating for my immunoprecipitation. The following is the information for my IP. Antibody for IP is rabbit anti- Fas receptor(M20) from santa cruz. Agarose G and rabbit IgG were used for preclear. For westernblot, primary ab is rat anti-fas ligand and secondary ab is goat anti-rat HRP. Tissue without IP antibody and only IP antibody without tissue were used as negative control. I actually got the bands what I want. There are 3 bands on my westernblot. I think two are IgG band and one is target band. But I also got the same bands on my negative control. I am wondering if somebody met the same problem.
Maybe you should IP with the monoclonal and western with the pAb? Also depending on what size your band of interest in it might be useful to cut away the heavy and light chain bands so that your blot will be cleaner ... then bands tend to "pop" when the "background" isn't blocking out your signal. Just my experience.
May I ask what do mean by' cut away the heavy and light chain bands'
Sorry for the stupid question.