Target protein is not detected in elution fraction despite successful capture on beads. Protein may remain bound to beads or antibody after elution attempt.
Common Causes
1Elution buffer pH is incorrect for disrupting antibody-antigen interaction (typically requires pH 2-3 or pH >10)
2Elution buffer ionic strength is insufficient to compete with antibody-antigen binding
3Wrong elution buffer type used (e.g., non-denaturing buffer when denaturing conditions required)
4Elution buffer concentration is too dilute to effectively disrupt protein complexes
Solutions
1Verify elution buffer pH matches protocol requirements; use glycine-HCl pH 2.5 for acidic elution or pH 11 for basic elution
2Increase elution buffer ionic strength by adding NaCl to 500 mM or using high-salt buffer formulations
3Switch to denaturing elution with SDS-PAGE loading buffer (2% SDS, 100 mM DTT) at 95°C for 5 min if appropriate
4Prepare fresh elution buffer at proper concentration and verify pH immediately before use