Western Blot Troubleshooting: High Background

Troubleshooting tips and solutions for high background in Western blot.

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High Background (Uniform Distribution)
Antibody concentration too high
  • Use higher antibody dilution.
Insufficient blocking
  • 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.

Inadequate washing
  • Increase washing time and volume.
Dry membrane
  • 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.

Sample degradation
  • 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: 

Positive controls:

Negative controls: