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Western Blot (Misshapen / Uneven Bands) severe

Uneven Bands Due to Improper Gel Polymerization

Symptom
Bands appear distorted, wonky, or uneven across lanes. In extreme cases, the same protein appears at different molecular weights in different lanes due to irregular gel matrix formation.
Common Causes
  1. 1 Incorrect TEMED concentration in gel recipe causing incomplete or uneven polymerization
  2. 2 Gel not fully covered with buffer during setting process
  3. 3 Uneven polymerization across gel thickness or width
  4. 4 Temperature fluctuations during gel polymerization phase
Solutions
  1. 1 Verify gel recipe and confirm correct TEMED volume is added
  2. 2 Ensure gel is completely covered with buffer during polymerization
  3. 3 Allow adequate polymerization time (minimum 30-45 minutes for resolving gel)
  4. 4 Prepare fresh acrylamide solutions and check ammonium persulfate (APS) activity
Related Video (3)
Bio-Rad Laboratories ★ 85
Setting Up and Running Mini-PROTEAN® TGX™ Precast Gels
"Demonstrates proper setup and execution of acrylamide gel casting and running, directly addressing gel polymerization protocols where TEMED concentration is critical."
Bio-Rad Laboratories ★ 78
Western Blotting
"Shows SDS-PAGE protein separation using Bio-Rad Western blot module, providing context for correct gel preparation and identifying how improper polymerization manifests as band distortion."
Bilibili (China-Accessible Mirrors) ★ 72
Western blot full protocol: Protein extraction to chemiluminescence
"Complete hands-on Western blot workflow including electrophoresis step, allowing observation of proper gel handling and identification of polymerization-related artifacts during protein separation."
Source: abcam.com ↗
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