Protein Complex Disruption in Co-IP

Symptom

In co-immunoprecipitation experiments, expected interacting proteins are not detected on Western blot, suggesting protein-protein complexes were disrupted during sample handling.

1. 1 Vigorous vortexing during sample processing disrupts weak protein-protein interactions
2. 2 Use of narrow pipette tips creates shear forces that dissociate protein complexes
3. 3 Excessive mechanical stress during washing or elution steps breaks non-covalent bonds in multi-protein complexes

1. 1 Vortex gently or use gentle rotation/rocking instead of vigorous vortexing during incubation and washing steps
2. 2 Use wide-bore pipette tips (cut tips if necessary) to minimize shear forces when transferring samples containing protein complexes
3. 3 Handle samples with minimal agitation to preserve weak non-covalent protein-protein interactions

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