Western Blot — Full Protocol Walkthrough (Bio-Techne)

critical

Complete Absence of Western Blot Signal

No protein bands are visible on the blot, even at the expected molecular weight position. Film or imaging system shows no detectable chemiluminescence or fluorescence.

💡 6 · ✓ 6

severe

High Background Signal on Western Blot

Overall high background across entire membrane reduces signal-to-noise ratio. Specific bands difficult to distinguish from background noise.

💡 6 · ✓ 6

severe

Weak or Diminished Western Blot Signal

Expected protein bands appear faint or barely visible on film or imaging system, even after extended exposure times.

💡 6 · ✓ 6

severe

High Non-Specific Background Signal

Entire membrane shows elevated signal intensity, making it difficult to distinguish specific protein bands from background noise. Signal-to-noise ratio is poor.

💡 6 · ✓ 6

severe

Weak or Faint Bands in Western Blot

Protein bands appear faint or barely visible on the Western blot membrane or film despite proper loading. Signal intensity is insufficient for detection or quantification.

💡 6 · ✓ 6

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

Reverse Image - White Bands on Dark Background

Film shows 'bleached' or 'burnt-out' white bands against dark background instead of expected dark bands. Bands appear as negative images where high protein concentration exists.

💡 3 · ✓ 3

moderate

Poor Detection of Small Molecular Weight Proteins

Proteins below 15-20 kDa show weak or absent signal while larger proteins are detected normally. Small protein bands appear diffuse or masked.

💡 4 · ✓ 4

moderate

Additional Bands or Wrong Molecular Weight

Blot shows unexpected extra bands at different molecular weights than predicted, or expected band appears at wrong size. Multiple bands may appear where single band is anticipated.

💡 4 · ✓ 4