No Signal Due to Stringent Lysis Buffer (Immunoprecipitation (CST Guide))

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Accepted Answer

No signal detected in co-immunoprecipitation experiment. Target protein appears absent despite expected expression. Common causes: 1) RIPA Buffer #9806 containing ionic detergent sodium deoxycholate disrupts protein-protein interactions 2) Stringent lysis conditions denature kinases and prevent protein complex formation 3) Inappropriate buffer selection for co-IP experiments destroys native protein associations 4) Insufficient sonication leads to incomplete nuclear rupture and DNA shearing, reducing protein recovery Solutions: 1) Use Cell Lysis Buffer #9803 instead of RIPA Buffer #9806 for IP and co-IP experiments 2) Perform sonication to ensure nuclear rupture, DNA shearing 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