Protein Complex Disruption in Co-IP (IP-Western Blot)

Question

Accepted Answer

In co-immunoprecipitation experiments, expected interacting proteins are not detected on Western blot, suggesting protein-protein complexes were disrupted during sample handling. Common causes: 1) Vigorous vortexing during sample processing disrupts weak protein-protein interactions 2) Use of narrow pipette tips creates shear forces that dissociate protein complexes 3) Excessive mechanical stress during washing or elution steps breaks non-covalent bonds in multi-protein complexes Solutions: 1) Vortex gently or use gentle rotation/rocking instead of vigorous vortexing during incubation and washing steps 2) Use wide-bore pipette tips (cut tips if necessary) to minimize shear forces when transferring samples containing protein complexes 3) Handle samples with minimal agitation to preserve weak non-covalent protein-protein interactions