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

Troubleshooting Guides

Download troubleshooting handbooks for IHC, Western blot and ELISA for FREE.

troubleshooting_box_image Troubleshooting guides

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 4oC. 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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Product Review

Anti-GRP94 Antibody (PA1340)

Review : "This is an excellent antibody to endoplasmin in HC11 cells. Clean, reliable detection with very little if any background. Great antibody for Westerns. Could probably be optimized for 1 hour primary incubation."

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