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

Causes:

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.

Causes:

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.

Causes:

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