Western Blotting Troubleshooting


Western Blotting

1. No Signal
• Confirm the amount of protein loaded and the quality of the protein sample.
• Check transfer efficiency.
• Check that the dilution of the primary is within the recommended range.
• Confirm that the primary antibody is compatible with the species being studied.
• Check the compatibility of the secondary antibody.
• Confirm that the secondary antibody and luminol reagents are functioning correctly.
• Analyse the antibody against known positive controls. 
2. High Background
• Check dilution and duration of incubation for the primary and secondary antibodies. If necessary, decrease incubation times from overnight at 4oC to 1 hour at room temperature.
• Check the blocking buffer used is 1x TBS, 5% Milk, 0.01% Tween-20 (Blotto A). If BSA is being used, change the buffer to Blotto A.
• Increase the blocking step to overnight at 4oC. Remember to omit Tween-20 from the buffer if blocking is conducted overnight.
• Check the secondary antibody is not causing the background by running the secondary antibody alone.
• If using PVDF, try switching to nitrocellulose. 
3. Non-Specific Bands
• Check the amount of protein loaded per lane, the dilution of the antibody being used and the duration of incubations with the primary and secondary reagents. Perform a serial titration of the primary and secondary antibodies to determine the optimal dilutions required and reduce the incubation time of the primary antibody with the membrane. If incubating overnight at 4oC, switch to 1 hour at room temperature
• Check non-specific binding of the secondary antibody by running a secondary-only control.
• Use the blocking peptide (where available) to identify between specific and non-specific bands. 
Have you any "tips" for us? We'd love to hear from you - the best will be posted on the website.