No Protein Detected: Insufficient Antibody Capture

Symptom

No target protein is detected in the elution fraction after immunoprecipitation procedure is completed. Western blot or other detection methods show no signal for the target protein.

1. 1 Antibody concentration is below the minimum required for effective capture of target protein
2. 2 Insufficient antibody amount used relative to the abundance of target protein in sample
3. 3 Antibody binding capacity exceeded by excess target protein without proportional antibody increase

1. 1 Check vendor datasheet for recommended antibody amount per IP reaction and verify current usage meets minimum
2. 2 Titrate antibody concentration upward in 2-fold increments (e.g., 2 µg, 4 µg, 8 µg) to optimize capture efficiency
3. 3 Calculate antibody:protein molar ratio and adjust antibody amount proportionally to sample protein concentration

Related Video (2)

Cell Signaling Technology ★ 85

Western Blotting Protocol

"Shows the complete Western blot workflow, which is the detection method for IP and helps troubleshoot missing signal."

Bio-Rad Laboratories ★ 70

Setting Up and Running Mini-PROTEAN® TGX™ Precast Gels

"Demonstrates gel electrophoresis setup used to separate and detect IP eluates, useful for checking the detection step."