No Bands with Recombinant or Overexpressed Protein

Symptom

No bands appear when testing recombinant protein or overexpression lysate, despite expected high protein levels, indicating structural or epitope accessibility issues.

1. 1 Truncated protein lacking antibody immunogen sequence region
2. 2 Immunogen located at same end as affinity tag, causing steric blocking of antibody recognition
3. 3 Incorrect protein amount or antibody dilution range not optimized for recombinant protein

1. 1 Select new antibody with immunogen located in the protein of interest region; contact vendor for immunogen details
2. 2 Redesign recombinant construct to position immunogen and tag at opposite ends; or use antibody with immunogen distant from tag
3. 3 Run protein amount panel with wide antibody dilution range to identify optimal detection conditions

Related Video (2)

Cell Signaling Technology ★ 85

Western Blot Troubleshooting Guide

"Direct troubleshooting guide addressing Western blot detection problems and diagnostic approaches to identify root causes like epitope accessibility issues."

Bilibili (China-Accessible Mirrors) ★ 72

Reliable and Reproducible Western Blot Results

"Demonstrates proper antibody incubation and detection methodology, providing context for understanding when truncated proteins fail to generate expected signal."