Troubleshooting for Western Blot

High Background
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:
· 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.