cell lysate for immunoprecipitation - (Feb/05/2008 )
Hello everyone, I have a question regarding sample of cell lysate to be used in immunoprecipitation. If cells are lysed let's say in RIPA buffer can I used these lysed samples directly into immunoprecipitation? What I'm basically asking is can the components of cell lysis buffer disrupt the antibody antigen and the antibody protein G interactions? If so what can be done to the sample before immunoprecipitation? Thanks hopefully ppl can understand my question.
RIPA should not affect the binding between your antigen and antibody. I always IP in lysis buffer and even wash beads with lysis buffer. If anything, it should give you a cleaner IP.
If you simply worry about antigen-Ab, then RIPA buffer shoud not be a problem. From the name of it, RIPA buffer is usually used for IP. But if you want to co-IP. looking for interacting proteins, then it depends on the nature of the pro-pro interacting between your protein (antigen) and its partners. The detergents in the lysis buffer might affect this interaction.