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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.
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.)
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.
Check for the presence of known isoforms in the literature or at uniprot.org.
Use an isoform-specific antibody.
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.
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: