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Western Blot (Sigma Protocol) severe

Incorrect Antibody Selection

Symptom
No signal or unexpected banding pattern despite proper protocol execution
Common Causes
  1. 1 Primary antibody not validated for western blotting application
  2. 2 Primary antibody cannot react with protein from the species being studied
  3. 3 Secondary antibody cannot bind to primary antibody from its species
Solutions
  1. 1 Verify antibody is validated for immunoblotting in product literature
  2. 2 Check antibody species reactivity matches your sample species
  3. 3 Test secondary antibody by spotting primary antibody on membrane and probing with secondary alone
  4. 4 Ensure secondary antibody is raised against the species of primary antibody
Related Video (3)
Cell Signaling Technology ★ 88
Western Blot Troubleshooting Guide
"A dedicated Western blot troubleshooting guide directly addresses symptoms like no signal or unexpected banding and antibody validation issues."
Bilibili (China-Accessible Mirrors) ★ 72
Western blot hands-on: Protein extraction through detection
"Hands-on protocol with explicit troubleshooting of common pitfalls, useful context for antibody selection and signal problems."
Bilibili (China-Accessible Mirrors) ★ 65
Western Blot Protocol Beginner Tutorial with Operations
"Beginner tutorial combining principles and operations that helps understand correct antibody use and expected workflow."
Source: sigmaaldrich.com ↗
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