Trouble Shooting

Western Blotting – Troubleshooting Problems and Solutions

Western Blot Troubleshooting

Common western blot problems with solutions.

Streaking of Western Blots

Causes: Over-loaded protein. Loaded too much protein or volume of sample into lanes / wells.

Solution: Load less protein quantity (volume) into wells and lanes.

Weak Bands or Weak Staining of Western Blots


A) Primary Antibody Conditions Poor:

  • 1° Antibody too dilute – increase 1° Antibody concentration
  • Insufficient 1° Antibody incubation time
  • Primary Antibody Old – stored in fridge too long use fresh 1° antibody
  • Repeated freeze and thaw cycles destroyed 1° antibody – use fresh 1° antibody

B) Secondary Antibody Conditions Poor:

  • Increase 2 ° Antibody time and concentration
  • Use fresh secondary antibody

Poor Transfer of Membranes

Symptoms: White / Clear areas on film where bands are supposed to be. Bubble-like clearing areas in film area. No Bands.

Causes: Poor Transfer Conditions such as:

  • Air Bubbles Between the Gel and the Western Blot Membrane
  • Transferred Proteins to the wrong side (i.e. you lost your proteins!)
  • Insufficient Current
  • Insufficient Transfer Time
  • Transfer Buffer poorly made
  • SDS concentration too high in Transfer Buffer

Dirty Blot

Symptoms: Blotchy Film. Black or Dark Areas on Film or membrane.


A) Poor Transfer Conditions such as:

  • Dirty Sponge or Transfer Apparatus
  • Used Transfer Buffer (proteins from previous transfers is getting transferred onto your membrane!)

B) Handling of Membrane with fingers / Dirty Membrane

C) Usage of Dirty Blotting Washing / Incubation Containers

Multiple Bands on Western Blot Membrane and Film

Symptoms: Many (larger or smaller) Bands where fewer were expected.


A) Antibody is cross-reactive to other proteins. Non-specific reactive to unrelated (non-specific) proteins.

  • Increase blocking / optimize western blot
  • Decrease primary antibody concentration

B) Samples and your protein of interest have been degraded proteolytically

  • especially when bands are smaller in size.

C) Aggregates of your protein or newly synthesized proteins of interest

  • very difficult to solve.

Western Blot