*Note to educators: you are permitted to share Boster Bio's resources and PDFs on your class websites and lab websites. Please make sure to cite or link to the origin.

Make sure you're prepared for the most common problems in Western Blotting. Troubleshooting eBooks and full protocols provided for Western Blot, ELISA and more!
BosterBio | Antibodies, ELISA Kits, Detection Kits
December 29, 2017
Dr. Booster
What's Your Western Blot Success Rate?

Greetings Earthling,

According to a report on GEN, 41% of researchers admit that their Western blots are unsuccessful at least 25% of the time.

Yikes! Western blotting (WB) is a widely practiced analytical technique to detect target proteins within samples using antigen-specific antibodies. When it fails to perform as expected, it can really be a downer.

common western blot issues

We’re here to help you succeed. Next time you encounter another problem with Western blot, we’ve compiled a checklist to help you troubleshoot your experiment.

Problem 1: High Background
Cause Solution
Antibody incubation temperature was too high
  • Incubate the antibody at a lower temperature, such as 4 o C. However, be aware that this may require a longer incubation time.
Antibody cross-reacted with other proteins or the blocking agent
  • Use a different blocking agent (Note: Do not use skim milk with the biotin system)
  • If non-specific secondary antibody binding is present:
    • Run the secondary antibody control (without the primary)
    • Decrease secondary antibody concentration
    • Test cross-reactivity between the secondary antibody & the membrane
Insufficient blocking
  • Extend the blocking time or use a compatible blocking agent (e.g. skim milk, BSA, serum, etc.)
Insufficient washing
  • Increase number of washes & buffer volume
  • Add 0.05% Tween 20 detergent into washing buffer
Problem 2: Weak/No Signal
Cause Solution
Insufficient sample loaded on the gel
  • Check the concentration of the protein samples
  • Load more protein
Loss of primary antibody effectiveness
  • Prepare fresh antibody and store properly when not in use
  • Avoid repeated freezing and thawing to minimize degradation
Inhibition of secondary antibody by sodium azide
  • Avoid adding sodium azide or using products containing sodium azide so that there is no interference with HRP-conjugated antibodies
Antigen masking by blocking buffer
  • Compare different blocking buffers
  • Optimize protein concentration of blocking agent
  • Reduce blocking time
Problem 3: Nonspecific Bands
Cause Solution
Primary antibody concentration was too high
  • Decrease primary antibody concentration
Excess protein on gel
  • Reduce amount of total protein loaded on gel
Insufficient washing
  • Increase number of washes
Blocking problem
  • Increase blocking time
  • Optimize choice of blocking agent
Access our Technical Resource Center for more WB tips and troubleshooting guidance.
Western Blot troubleshooting ebook guide download PDF

*Note to educators: you are encouraged to share BosterBio's resources and PDFs on your class and lab websites, please cite or link to origin bosterbio.com

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