Lysis of Cultured Cells for Immunoprecipitation
Adapted from Proteins and Proteomics, by Richard J. Simpson. CSHL Press, Cold Spring Harbor, NY, USA, 2003.INTRODUCTION
Cell lysis with mild detergent is commonly used with cultured animal cells. If low detergent concentrations are sufficient to cause cell lysis (e.g., 1% Nonidet P-40 [NP-40] or 1% Triton X-100), this method can be gentler to the protein of interest than mechanical homogenization methods. The choice of detergent must be tailored to the nature of the epitope recognized by the immunoprecipitating antibody. If the antibody recognizes a linear peptide epitope (e.g., a synthetic peptide), then use a harsh denaturing lysis buffer (e.g., RIPA buffer). On the other hand, if the antibody is directed toward a conformational epitope, use NP-40 lysis buffer (or 1% Triton X-100). This protocol presents separate methods for lysing cells grown as monolayer cultures and for cells grown in suspension.
