No Signal in Co-IP Due to Stringent Lysis Buffer (Immunoprecipitation (CST Guide))

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No signal detected in co-immunoprecipitation experiment despite adequate protein expression confirmed by input lysate control. Western blot shows no target protein bands after IP. Common causes: 1) RIPA Buffer (#9806) containing ionic detergent sodium deoxycholate disrupts protein-protein interactions 2) Kinase denaturation caused by denaturing buffer components incompatible with co-IP 3) Insufficient sonication leading to incomplete nuclear rupture and poor protein recovery 4) Membrane protein complexes disrupted by overly stringent detergent conditions Solutions: 1) Use Cell Lysis Buffer #9803 instead of RIPA Buffer for all IP and co-IP experiments 2) Perform adequate sonication to ensure nuclear rupture, DNA shearing, and maximal protein recovery without disrupting protein complexes 3) Include input lysate control to verify target protein expression and antibody functionality 4) Probe blot with antibody to IP protein to confirm successful pull-down of primary protein