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

Temperature-Dependent Sample Degradation

Protein degradation, dephosphorylation, or denaturation occurs when samples are kept above 4°C throughout the protocol, resulting in high background or loss of target bands.

💡 3 · ✓ 3

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

No Signal or Weak Signal

No bands or very faint bands appear on the western blot after detection

💡 5 · ✓ 5

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

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

Weak or Absent Signal Due to Insufficient Exposure

Western blot film shows very faint bands or no visible bands after initial exposure. The membrane may contain protein and antibody binding has occurred, but the chemiluminescent or colorimetric signal is not captured adequately on film.

💡 4 · ✓ 4

moderate

Electrophoresis Time Not Optimized for Target Protein

Protein bands do not align with expected molecular weight markers, with systematic upward or downward shifts affecting all bands uniformly. Pre-staining reveals migration issues before antibody detection.

💡 4 · ✓ 5

moderate

Inactive Chemiluminescent Detection

No signal detected when using chemiluminescent detection method despite proper procedure

💡 3 · ✓ 3

moderate

Weak bands (faint signal)

Bands are visible but barely detectable; long exposure times are needed, and the internal control also appears weak.

💡 5 · ✓ 5