High Background from Antigen Degradation During IP

Symptom

High background with smeared or degraded protein bands in IP eluate, often accompanied by loss of target protein signal. Multiple lower molecular weight bands appear below the expected target band.

1. 1 Protease inhibitors not added to lysis buffer or have lost activity
2. 2 Protease inhibitors added too late after cell lysis has begun
3. 3 Sample kept at room temperature too long during IP procedure
4. 4 Old or improperly stored protease inhibitor stock solutions used

1. 1 Add fresh protease inhibitors immediately when lysing samples, before mechanical disruption
2. 2 Prepare protease inhibitor cocktail fresh or use single-use aliquots stored at -20°C
3. 3 Keep samples on ice throughout the entire IP procedure
4. 4 Complete IP procedure within 4-6 hours and store lysates at -80°C if not used immediately

Related Video (2)

Cell Signaling Technology ★ 80

Western Blotting Protocol

"Western blot protocol is directly downstream of IP and shows how to handle protein samples, but does not specifically address protease inhibitor usage."

Bio-Rad Laboratories ★ 55

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

"Shows gel running and sample handling, tangentially related to detecting degraded bands, but not specific to IP or protease inhibitors."