No Bands Troubleshooting in Western Blots

Why are there no bands in a western blot film? This Western Blot troubleshooting guide has listed the possible reasons and relative solutions for no western blotting signal.

Western Blot possible causes & solutions for no bands

No bands in a western blot

No bands in a western blot
Possible cause Solution
Not enough transferred protein • The protein expression level may be too low, so just increase the volume of loaded protein;
• Use a positive control, and make sure that the lysis buffer you used for sample preparation was strong enough to break the cell wall or membrane, and have included appropriate protease inhibitors;
• Check that if the separated proteins have successfully transferred to the membrane by ponceau staining.
Too low antibody concentration • Reduce the dilution proportion of primary antibody or secondary antibody; quit the re-used antibody and try a fresh one.
Antibody incompatibility • Ensure that the primary antibody you used can well recognize your target protein in the species being analyzed by performing a Clustalw alignment, and also, the secondary antibody must be chosen according to the primary antibody. e.g. if the primary antibody is a rabbit monoclonal antibody, you should use an anti-rabbit secondary antibody.
Over wash of the membrane • Avoid excessive washing of the membrane.
Too much blocking • Excessive blocking makes it difficult to visualize your target protein, so reduce the concentration of non fat milk appropriately or shorten the blocking time.
Sodium azide • Remove sodium azide from all buffers as HRP-conjugated secondary antibodies will be inhibited by sodium azide.
Inactive substrate • Switch to fresh substrate.
Not enough film exposure • Increase the time of film exposure.
Small proteins migrating through the transfer membrane •Choose the membrane type 0.2 µm instead of 0.45 µm; reduce transfer time or current.