Western Blot Troubleshooting: High Background

Troubleshooting tips and solutions for high background in Western blot.

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.

Proteintech control antibodies are $189 each for a 150ul size vial

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:

• Samples from cells overexpressing the target protein.
• Purified recombinant protein.
• Cell line/tissue with proven positive signal. 

Negative controls:

• Samples targeted with RNA interference.
• Samples from knockout tissues/cells.
• Cell line/tissue with proven negative signal.