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
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.