protein

Co-IPs are a highly specialized approach to identifying in vivo protein:protein interactions. The protein preparation is fairly similar to a total protein prep, however, a different buffer is used for lysis, and a column in used to run the fraction through. Our lab uses micro-max columns which consist of thousands individual iron atoms fused to a primary antibody (typically C-myc or HA). Running the intial fraction through this column will grab the input, or protein complex that you are initially probing, in other words, one of the two anticipated interactors. This fraction is quantified/normalized to other samples that are later run out via SDS-PAGE. This is then transferred to a charged membrane that can be probed with a different specific antibodies, which would detect the secondary interacting protein. It is crucial to rinse or wash the membrane between probing steps to eliminate any risidual or background primary antibody that has unspecifically bound to the membrane.