2.11. Immunoprecipitation Assay

Cells were lysed with immunoprecipitation (IP) lysis buffer (20 mM HEPES, pH7.9; 1.5 mM MgCl₂; 0.4M NaCl; 0.5 mM EDTA; 1% NP-40; 10% Glycerol; 1 mM DTT; 1 mM PMSF; 1X protease inhibitor cocktail). Cell lysates were pre-cleared with protein A/G agarose beads, followed by immunoprecipitation with the appropriate antibodies at 4°C overnight in the presence of 20 μl of protein A/G agarose beads. The beads were washed three times with lysis buffer, resuspended in 20 μl of 2X loading buffer and then subjected to immunoblot analysis.