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.

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

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 ★ 78

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

"Shows proper setup and running of acrylamide gels, the exact context where TEMED/polymerization issues cause uneven band migration."

Cell Signaling Technology ★ 72

Western Blotting Protocol

"Full Western blot protocol emphasizing reproducibility, useful for learning correct gel casting and troubleshooting distorted bands."

Bilibili (China-Accessible Mirrors) ★ 70

Western blot hands-on: Protein extraction through detection

"Hands-on WB protocol with explicit troubleshooting of common pitfalls, directly relevant to diagnosing uneven bands."