Western Blot Uneven Signal: Causes and Fixes
Uneven signal across lanes is a common problem that makes quantification difficult and results unreliable. This guide explains why signals are uneven, provides diagnostic steps, and offers expert solutions to ensure consistent signal intensity across all lanes.
Common Causes of Uneven Signal
Sample-Related Causes
- Unequal protein loading
- Inconsistent sample preparation
- Variable protein concentration
- Sample degradation differences
Technical Causes
- Uneven transfer
- Inconsistent antibody incubation
- Uneven substrate application
- Membrane handling issues
Diagnostic Steps
How to Diagnose Uneven Signal
- Check Ponceau S staining to verify equal protein loading
- Compare loading control (actin, GAPDH) across lanes
- Verify equal sample volumes were loaded
- Check for uneven transfer by staining gel after transfer
- Inspect membrane for bubbles or contact issues
- Verify antibody incubation was even
Solutions
Fix Sample Loading
- Quantify all samples accurately before loading
- Load equal amounts of protein (e.g., 20 μg per lane)
- Use consistent sample buffer volumes
- Normalize samples to same concentration
Fix Transfer Issues
- Ensure good contact between gel and membrane
- Remove all bubbles during transfer setup
- Use consistent transfer conditions
- Verify transfer efficiency with Ponceau S
Fix Detection Issues
- Apply detection substrate evenly across membrane
- Ensure membrane is flat during detection
- Use consistent exposure settings
- Image immediately after detection
Prevention Tips
- Always quantify samples before loading
- Use loading controls to verify equal loading
- Maintain consistent protocol across all samples
- Handle membrane carefully to avoid damage
- Ensure even antibody and substrate distribution