Western Blot Full Protocol Video Version

severe

Protein concentration is too low

Nanodrop reads A280 ≈ 0.09, concentration ≈ 0.05 mg/mL; SDS-PAGE shows very faint overall bands; BCA quantification confirms low yield.

💡 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

severe

Alkaline Phosphatase Inhibition by Tween 20

Western blot using alkaline phosphatase (AP)-conjugated antibodies shows weak or no colorimetric signal development. The BCIP/NBT or other AP substrates fail to produce expected colored precipitate even with adequate incubation time.

💡 4 · ✓ 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

Low Molecular Weight Protein Signal Loss

Proteins smaller than 25-30 kDa show weak or absent signal despite adequate expression levels. Higher molecular weight proteins from the same sample transfer successfully.

💡 3 · ✓ 4