Western Blot No Signal Troubleshooting Guide
No signal in western blot requires systematic troubleshooting. This comprehensive guide provides diagnostic flowcharts, common causes, and proven solutions to help you identify and fix the problem quickly.
Diagnostic Flowchart
- Check positive control: Does positive control show signal? If no, problem is in detection system.
- Verify transfer: Use Ponceau S to check if proteins transferred to membrane.
- Check sample: Does sample contain target protein? Use known positive sample.
- Verify antibody: Test antibody with positive control sample.
- Check detection: Verify secondary antibody and detection substrate are working.
Sample Diagnostics
Check Protein Presence
- Use positive control sample known to contain target protein
- Verify protein expression in your sample (other methods)
- Check sample preparation - ensure proper extraction
- Verify protein concentration is sufficient
Sample Loading Issues
- Increase loading amount to 50-100 μg
- Check for protein degradation
- Verify sample buffer composition
- Ensure equal loading across lanes
Antibody Diagnostics
Antibody Testing Steps
- Test antibody with positive control sample
- Try different antibody dilutions (1:100 to 1:5000)
- Extend incubation time (overnight at 4°C)
- Check antibody expiration date
- Verify antibody specificity for your protein
- Test different antibody batches if available
Transfer Diagnostics
Transfer Verification
- Stain membrane with Ponceau S to visualize proteins
- Check for complete transfer - no protein in gel
- Verify transfer conditions (time, voltage, buffer)
- Check membrane activation (PVDF needs methanol)
- Ensure proper gel-membrane contact
Detection Diagnostics
Secondary Antibody
- Verify matches primary antibody species
- Check dilution (typically 1:5000)
- Test with positive control
- Check expiration date
Detection Substrate
- Check expiration date
- Use fresh substrate
- Try enhanced ECL substrates
- Optimize exposure time
Systematic Troubleshooting Approach
- Start with positive control: Verify your detection system works
- Check transfer: Ensure proteins transferred to membrane
- Verify sample: Confirm sample contains target protein
- Test antibody: Verify antibody works with positive control
- Optimize conditions: Adjust dilutions and incubation times
- Enhance detection: Use sensitive substrates and optimize exposure