Antibody concentration too high
Use higher antibody dilution.
· Increase the concentration of blocking reagent (e.g., from 5 to 7%).
· Increase blocking time and/or temperature.
· Add Tween 20 to the blocking buffer.
· Include blocking reagent and Tween 20 in the primary antibody dilution buffer.
Increase washing time and volume.
Ensure membrane does not dry out during the immunoblotting process.
Non-specific binding of secondary antibody
· Perform a secondary antibody-only control experiment (omit the primary incubation step).
· Use a pre-adsorbed secondary antibody with reduced crossreactivity to unwanted species.
· For phosphorylated protein detection, do not use milk-based buffers such as non-fat milk or casein buffer. (Milk and casein are phosphoprotein-rich.)
Film exposure too long / Detection reagent too sensitive
Check different types and dilution of the detection reagent.
High Background (Non-Specific Bands )
The troubleshooting tips for high background (uniform distribution) can also be applied to scenarios where non-specific but distinct bands appear on the Western blot membrane.
The following should also be considered:
Target protein abundance is lower than the threshold of nonspecific binding
· Load more protein per well at SDS-PAGE.
· Enrich low-abundance proteins by immunoprecipitation, fractionation, etc.
· Prepare fresh lysates each time
· Use freshly prepared sample kept on ice up until the addition of sample buffer and immediate heating to 95°C for 5–10 minutes.
· Tissue extracts tend to produce more non-specific bands and degradation products. Use fresh, sonicated, and clarified tissue extracts.
· Always include protease inhibitors (and phosphatase inhibitors for the detection of phosphorylated targets).
· Ensure sample has not degraded.
Interference from other isoforms
· Check for the presence of known isoforms in the literature or at uniprot.org.
· Use an isoform-specific antibody.
Inefficient SDS-PAGE separation
· Change the gel percentage to suit the target protein’s MW.
· Lower percentage Tris-Glycine gels should be used for larger proteins, or use Tris-Acetate-based gels and buffers. (See page 12 for our SDS-PAGE gel recipes).
· Higher percentage Tris-Glycine gels (up to 15%) should be used for smaller proteins (<20 kDa) or use Tris-Tricine gels.
Presence of post-translational modifications
Know your protein of interest, band sizes can shift due to glycosylation, phosphorylation, precursor maturation, etc.
Lack of controls
While the omission of control samples from a Western blot is not a cause of non-specific bands, their inclusion can tell you why you may be seeing them on your membrane. Controls you could include are:
· Samples from cells overexpressing the target protein.
· Purified recombinant protein.
· Cell line/tissue with proven positive signal.
· Samples targeted with RNA interference.
· Samples from knockout tissues/cells.
· Cell line/tissue with proven negative signal.