Western Blot Troubleshooting: High Background
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- Western Blot Protocol
- How To Optimize Your Western Blot
- SDS-PAGE Gel Recipes
- How To Optimize Your Results With Low MW Proteins
- Tricine Gel Recipe For Low Molecular Weight Proteins
- Choosing The Right Lysis Buffer
- Choosing The Right Western Blot Detection Method
- Western Blot Troubleshooting: Why Does The Observed Protein Molecular Weight (MW) Differ From The Calculated One?
- Western Blot Troubleshooting: High Background
- Western Blot Troubleshooting: Weak/No Signal & Other
- Western Blot ppt
- Western Blot Video Protocol
High Background (Uniform Distribution)
Antibody concentration too high
- Use higher antibody dilution.
Insufficient blocking
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Increase the concentration of blocking reagent (e.g., from 5 to 7%).
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Increase blocking time and/or temperature.
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Add Tween 20 to the blocking buffer.
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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
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Perform a secondary antibody-only control experiment (omit the primary incubation step).
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Use a pre-adsorbed secondary antibody with reduced crossreactivity to unwanted species.
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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 following should also be considered:
Target protein abundance is lower than the threshold of nonspecific binding
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Load more protein per well at SDS-PAGE.
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Enrich low-abundance proteins by immunoprecipitation, fractionation, etc.
Sample degradation
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Prepare fresh lysates each time
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Use freshly prepared sample kept on ice up until the addition of sample buffer and immediate heating to 95°C for 5–10 minutes.
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Tissue extracts tend to produce more non-specific bands and degradation products. Use fresh, sonicated, and clarified tissue extracts.
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Always include protease inhibitors (and phosphatase inhibitors for the detection of phosphorylated targets).
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Ensure sample has not degraded.
Interference from other isoforms
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Check for the presence of known isoforms in the literature or at uniprot.org.
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Use an isoform-specific antibody.
Inefficient SDS-PAGE separation
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Change the gel percentage to suit the target protein’s MW.
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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). •
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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:
