Stringent wash buffer for Immunoprecipitation - What decides stringency of wash buffer when perform IP? (May/02/2007 )

Hi folks,

I am doing IP (for flag-tagged protein) using a-flag antibody, but I have some background bands in control cell lysate which is not supposed to have any flag-protein when I stained the SDS-PAGE gel. So my friend suggested me to wash bead-antibody-protein complex with more stringent wash buffer.

Right now I am using cell lysis buffer (ripa) with 1% of NP-40 for wash. Don't you think this is stringent enough? What else can I change to increase stringeny of wash buffer?

Thanks in advance.

increase NaCl concentration up to 500 mM

Do you think 1% NP-40 is enough?