Multiple Bands Troubleshooting in Western Blots

The Western blot assay is a powerful tool to study a protein of interest. However, if multiple bands appear in a Western Blotting, this may increase difficulties and troubles in the protein analysis. Also, one should determine the reason of multiple bands, because they can be caused by technical artifacts or represent true variants of the protein of interest.

The following Western Blot troubleshooting guide are based on our many years of experience, and can help you solve multiple bands / too many bands issues in Western Blottings.

Western Blot possible causes & solutions for multiple bands

Multiple bands/too many bands  in a western blot

Multiple bands / too many bands in a Western Blot
Possible cause Solution
non-specific binding of primary or secondary antibodies. • Use 2% non-fat dry milk in blotting buffer as a starting point so as to dilute primary and secondary antibodies. Adjust antibody concentration down or up as needed.
• Add 0.1 - 0.5% Tween 20 to primary or secondary antibody solution.
• Increase NaCl concentration in blotting buffer used for antibody dilution and washing steps (the recommended range 0.15 M - 0.5 M).
• Increase Tween 20 concentration in blotting buffer used for washing steps (0.1%-0.5%). Increase times of washing steps.
An excessive amount of lysate loaded onto the gel. • Using decreasing dilutions of lysate will help to determine optimal loading amounts. An increased wash cycle may also alleviate this problem.
Lower molecular weight products may be due to protein degradation. • Prepare fresh samples by lysing cells/tissues according to our recommended protocol. Compare the signal of your experimental samples with the positive control.
Post-translational protein modifications. • Higher molecular weight products may be a result of post-translational protein modifications, including, but not limited to, glycosylation, myristylation, phosphorylation, and/or ubiquitination.
Protein aggregation. • Higher molecular weight bands may also be a result of protein aggregation that is not resolvable by SDS and boiling.
Proteolytic breakdown of the antigen. • This is not uncommon, particularly if samples are stored for prolonged time or if proteins or membranes are fractionated after homogenization of the starting tissue. All additional bands are of lower apparent molecular mass than the full-length protein. Particularly susceptible are synapsins and synaptotagmins. Addition of protease inhibitors such as PMSF, pepstatin or leupeptin should be considered.
Ineffecient blocking. • A variety of different blocking agents are described in the literature including nonionic detergents and/or proteins. Change of the blocking conditions may remedy the problem.
Concentration of antigen too low. • The resolution of SDS-PAGE is limited to 50 -100 bands. If the relative concentration of the antigen of interest is too low (less than 0.2% of total protein), it may be difficult to detect. For instance, synaptobrevin/VAMP comigrates with histones in cell homogenates which interfere with its detection. Signal enhancement may then lead to the appearance of artificial bands. Enrichment of the antigen by fractionation or by immunoprecipitation should be considered.