Western Blot No Signal After Transfer: How to Fix
No signal after transfer can be frustrating, especially when you've confirmed that proteins transferred to the membrane. This guide provides systematic troubleshooting steps to identify and fix the cause of no signal after successful transfer, covering antibody issues, detection problems, and protocol errors.
Step 1: Verify Transfer Was Successful
Transfer Verification Methods
- Stain membrane with Ponceau S to visualize transferred proteins
- Check for pre-stained ladder bands on membrane
- Stain gel after transfer with Coomassie Blue to check for residual protein
- Verify transfer efficiency is >80%
Step 2: Check Antibody Issues
Primary Antibody
- Verify antibody specificity and expiration date
- Check antibody concentration (may be too low)
- Confirm antibody is correct for your target protein
- Test with positive control sample
Secondary Antibody
- Verify secondary antibody matches primary antibody species
- Check secondary antibody concentration
- Confirm secondary antibody is active
Step 3: Check Detection System
- Verify detection substrate is fresh and not expired
- Test detection reagents on clean membrane
- Check imaging system is working correctly
- Verify exposure time is appropriate
- Test with positive control blot
Common Causes
- Antibody concentration too low
- Antibody expired or inactive
- Wrong antibody species or specificity
- Detection substrate expired
- Insufficient blocking
- Excessive washing
- Incorrect detection conditions
Solutions
- Increase primary antibody concentration (try 1:500 to 1:1000)
- Extend primary antibody incubation to overnight at 4°C
- Use fresh detection substrate
- Test with positive control to verify system
- Optimize blocking conditions
- Reduce washing stringency
- Verify all reagents are fresh and active