Western blot full protocol: Protein extraction to chemiluminescence

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

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

Multiple Low Molecular Weight Bands or Smearing Below Expected Band

Multiple bands appear at molecular weights lower than the expected target protein, often accompanied by smearing or streaking below the primary band. This pattern suggests progressive breakdown of the intact protein.

💡 4 · ✓ 4

severe

White Bands or Ghost Bands with ECL Detection

Target protein bands appear white or light-colored instead of dark on film or imager. The bands are present but show inverted contrast, appearing as 'ghost bands' where signal should be strongest.

💡 5 · ✓ 6

severe

Insufficient Protein Sample Loading

Western blot shows weak or no signal for target protein. Loading controls may also appear faint, indicating insufficient total protein loaded per lane. This is particularly problematic for low-abundance target proteins.

💡 4 · ✓ 5

severe

White Spots or Smudges on Western Blot Membrane

Uneven white spots, circular patches, or smudged areas appear on the membrane where no signal is detected. These areas correspond to regions where protein transfer did not occur.

💡 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

Dark or Black Blot with Ghost Bands

Entire membrane appears dark or black after chemiluminescent detection, often with white 'ghost' bands where protein bands should appear. Background signal is saturated across the whole blot.

💡 4 · ✓ 5

moderate

Reverse Image: White Bands on Dark Background

Film shows negative or reverse image with white or 'bleached' bands on dark background instead of expected dark bands. Also called 'burnt-out' bands.

💡 3 · ✓ 4

moderate

Inactive Chemiluminescent Detection

No signal detected when using chemiluminescent detection method despite proper procedure

💡 3 · ✓ 3

moderate

Weak Lysis Method Causing Nonspecific Bands

Insufficient lysis strength results in incomplete protein extraction, leading to weak target signal and increased nonspecific bands from partially solubilized proteins.

💡 3 · ✓ 3

moderate

Non-Specific Signal from Insoluble Protein in Well or Dye Front

Strong signal appears at the top of the gel (in or near the loading well) or at the bottom (dye front), unrelated to the expected molecular weight of the target protein. This is especially common for very high molecular weight proteins (>250 kDa).

💡 4 · ✓ 5

minor

Blue Background on Gel or Blot After Electrophoresis

After completing electrophoresis, the gel or blot membrane displays a diffuse blue background coloration instead of a clear background. The blue tint is distributed across the gel surface or transferred to the membrane.

💡 4 · ✓ 4