Western Blot Full Protocol Video Version

critical

Severe protein degradation

SDS-PAGE shows obvious low-MW degradation bands and a smear/tail. Target band is faint or absent on Western.

💡 5 · ✓ 5

severe

Incomplete lysis — visible debris or turbid supernatant

After homogenization / lysis there are visible chunks or debris in the tube and the supernatant is turbid; SDS-PAGE shows missing or weak bands.

💡 5 · ✓ 5

moderate

Viscous sample — stringy lysate, hard to pipette

Lysate is stringy/sticky, pipettes slowly, sticks to the tip, and gives unreliable BCA quantification and bad SDS-PAGE loading.

💡 4 · ✓ 4

moderate

Inadequate Antibody Incubation Time

Western blot yields weak signal despite using appropriate antibody concentrations and sample amounts. The antibody-antigen binding has not reached equilibrium, resulting in suboptimal signal development.

💡 4 · ✓ 4

moderate

Extra Bands at 50 kDa and 25 kDa Masking Target

Western blot shows strong bands at approximately 50 kDa (heavy chain) and 25 kDa (light chain) that may mask or interfere with the target protein band of interest.

💡 3 · ✓ 3