Troubleshooting for Western Blot

Western Blot (WB) is one of the most used techniques in proteomics. Many factors can significantly affect the results. Therefore, Wb methods often require a lot of optimization and troubleshooting to ensure the accuracy of the experimental results. Common problems with WB cover no signal, high background, special bands. Creative Biolabs provides global clients with typical WB problem solving tips and advice to help you solve specific WB challenges.

Here we list the common problems of WB, and analyze the possible causes and give the corresponding solutions.

Low or No Signal

  1. Proteins were washed from the membrane during assays.
    • Reduce the duration or number of washing steps and the stringency of washing conditions.
    • Reduce washing temperature.
    • Reduce the concentration of detergent.
  2. Insufficient antibody binding to protein.
    • Use a higher concentration of primary antibody.
    • Incubate samples with antibodies overnight at 4°C.
  3. Low antigen-antibody binding affinity.
    • Reduce the number and time of washes.
    • Increase antibody concentration.
  4. Incompatible primary and secondary antibodies.
    • Use secondary antibodies raised against the primary antibody species.
    • Make sure that the primary and secondary isotypes are compatible.
  5. The exposure time was insufficient.
    • Try to optimize the exposure time to image the blot again.
  6. Diluted antibody for reuse.
    • Use freshly diluted antibodies to ensure experimental results.
  7. Protein is absent or low in content.
    • Check the literature to ensure protein content.
    • Make sure there is enough protein in each lane, use protease inhibitors, and run a positive control.

High Background

  1. The secondary antibody may be binding non-specifically.
    • Run a control without any primary antibody.
    • Use the proven secondary antibody.
  2. Too much antibody.
    • Dilute antibodies to optimal concentration.
  3. Not enough washing between steps.
    • Increase the number and time of wash steps.
    • Increase wash buffer volume.
  4. Incomplete blocking of the membrane.
    • Increase the duration of the blocking step.
    • Use different blocking agents.
  5. Long exposure time.
    • Use a shorter exposure time.

Multiple or Unexpected Bands or Non-specific Binding

  1. The protein may have multiple isoforms.
    • Check the literature to see if more than one isoform is reported.
    • Check in UniProt to see if multiple isoform sequences are listed.
  2. Post-translational modifications (PTMs).
    • PTMs can result in multiple bands on WB. For example, glycosylation, SUMOylation, ubiquitination, and phosphorylation can cause multiple bands to appear on WB. After treatment of the sample with a suitable reagent to break down the modified protein, the additional band disappears.
  3. The cell line may have been passaged too many times.
    • Prepare original non-passaged cell line for experiments.
  4. The antibodies are not purified.
    • Use affinity purified antibody.

Unusual Gel or Band Appearance

  1. The voltage during the experiment is too high.
    • Use the appropriate voltage and lower it if necessary.
  2. The gel has polymerized unevenly.
    • Make sure to add the appropriate amount of TEMED and mix well. The gel was completely covered with buffer as it solidified.
  3. Gel overheating.
    • Run the gel at a low temperature. Place the electrophoresis tank on ice or in a cooler at 4°C during the run.

For many years, Creative Biolabs is happy to provide advanced technical support and expert troubleshooting tips to help clients resolve WB problems. Based on an optimized technology platform, we are committed to providing a variety of natural autoantibodies (NAA) services. Please do not hesitate to contact us for more information and a detailed quote.

For Research Use Only | Not For Clinical Use

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