Tips and Tricks for troubleshooting common western blot issues

Nov 17, 2023

Western blots are a ubiquitous technique used in research labs to study proteins of interest. Western blotting is used for antibody validation, protein-protein interaction studies, and many other applications.¹ However, generating interpretable western blot results can be challenging. Below are some commonly encountered issues and how to resolve them.²

High Background

Could be caused by	Try this
High antibody concentration	Set up a titration with both the primary and secondary antibody. Include a known protein control.
Blocking solution	Use fresh blocking solution, increase blocking time/temperature, try different blocking agents.
Blot dried out during blocking/washes	Ensure membrane is adequately covered during blocking and wash steps.
Not enough washes	Increase the number of washes and the volume of wash solution. Ensure a minimum of 3 washes of 5 min each.
Over-exposure of film	Try a shorter exposure.

No Signal

Could be caused by	Try this
Antibody concentration too low	Set up a titration with both the primary and secondary antibody. Include a known protein control.
Low amounts of target in sample	Increase protein concentration on gel. Titration is recommended.
Primary and secondary antibody mismatch	Ensure secondary antibody is against the host species that the primary was raised in.
Problems with transfer	Use protein markers with color to check transfer. Optimize transfer using control protein. Check for air bubbles before transfer.
Too many washes	Reduce number of washes.

Multiple Bands

Could be caused by	Try this
Gel overloaded	Titrate protein samples loaded on gel.
Too much antibody used	Set up a titration with both the primary and secondary antibody. Include a known protein control.
Over-exposure of film	Try a shorter exposure.
Issues with blocking	Use fresh blocking solution, increase blocking time/temperature, try different blocking agent.
Post-translational modification of target protein	Research published examples of post-translational modifications of target protein which could provide a biological explanation (i.e., phosphorylation, glycosylation, ribosylation).
Post-translation cleavage	Many proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. pro-caspases. Research published examples for your target.
Splice variants	Alternative splicing may create different sized proteins produced from the same gene. Research published examples for your target.

OriGene solutions for western blotting:

• Recombinant proteins – use as controls, or for assay development
• Over-expression Lysates – use as controls, or for assay development
• Primary antibodies
• Secondary antibodies
• Anti-tag antibodies

For questions, contact Technical Support at techsupport@origene.com

References

1. https://www.technologynetworks.com/analysis/articles/western-blot-procedures-analysis-and-purpose-353918
2. https://www.news-medical.net/whitepaper/20200128/A-Guide-to-Solving-Western-Blot-Protocol-Issues.aspx