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

16,000+ Antibodies, find yours: Shop now!

Buy Primary Antibody Get Secondary FREE

Buy Primary Antibody Get Secondary FREE

Boster Products for Flow Cytometry

Boster Guarantee

All Boster antibodies and ELISA kits are guaranteed to meet the specifications on the data sheet. We promise to thoroughly investigate any concerns about the quality of our products; if you encounter a problem with a Boster antibody or ELISA kit, our technical support team will respond with personalized advice within 24 hours. If we can’t make your experiment work, we will refund your purchase in full or provide a replacement free of charge.

100% Satisfaction Guaranteed

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."

Read More

About Boster Biological Technology

Here are some quick facts about bosterbio.com

26
Years

2300+
Publications

4.8 / 5
Rating on biocompare.com

16000+
Antibodies

900+
ELISA Kits