A Guide to Solving Western Blot Protocol Issues

At some point in time most people will have difficulty in getting a western blot protocol to work. This western blot troubleshooting guide is designed to help target the potential cause and test out solutions.

Problem: Weak or No Signal

Potential Cause: Incorrect antibody concentration

  • Use a higher concentration of antibody, or a longer incubation
  • Both primary and secondary antibodies should be optimized with every experiment

Potential Cause: Primary antibody does not recognize the antigen

  • Verify that the antibody is definitely able to detect the antigen in the species by checking data sheets and reference material

Potential Cause: Primary and secondary antibody mismatch

  • Raise the secondary antibody against the host species of the primary antibody. For example, if the primary was raised in mouse (i.e. Mouse Anti-HSP70) an anti-mouse should be used for the secondary (i.e. Goat Anti-Mouse)

Potential Cause: Storage issues

  • The antibody structure can degrade with freeze/thaw cycles. Aliquot antibodies into one time use vials upon delivery.
  • If the antibody was not stored in line with recommendations, then unfortunately a new vial might need to be used instead.

Potential Cause: Antigen level is too low

  • Ensure that sufficient protein is loaded onto the gel (~20 µg)
  • It is sometimes necessary to induce cells in order to produce more protein before harvest.
  • Make sure that the protein is not degraded

Potential Cause: Protein is not highly expressed in the cells/tissue

  • Isolate the cellular compartment containing the protein of interest (i.e. mitochondria or cellular membrane) in order to concentrate the protein lysates.
  • Try to use ECL western blot as opposed to the colorimetric western blot.

Potential Cause: Epitope is masked by the blocker

  • Try a different blocker agent
  • Try optimizing the concentration of the blocker

Potential Cause: Protein did not transfer

  • Optimize the transfer protocol for the specific protein

Potential Cause: Secondary antibody tag not visible

  • Make sure that the secondary antibody tag was not exposed to excessive light.
  • Use a longer incubation time

Potential Cause: Excessive Washing

  • Reduce the amount of washing steps

Problem: High Background Staining

Potential Cause: High Antibody Concentration

  • Try reducing the concentration of primary and/or secondary antibody
  • Both the primary and secondary antibodies should be optimized with every experiment

Potential Cause: Insufficient Blocking

  • Try increasing the blocker incubation time and/or the temperature
  • Try increasing the concentration of blocker
  • Use a different blocker such as BSA, casein, or milk
  • The recommendation is 5% w/v non-fat dry milk in Tris buffered saline with 0.05% TritonX100, TBST overnight at 4 oC or 1 hour at RT.

Potential Cause: Insufficient Washing

  • Increase the volume and amount of washes
  • Increase the concentration of detergent in wash buffer (i.e. 0.05% Tween-20)

Potential Cause: Contaminated Solutions

  • Potential contaminants should be kept in mind. Clean equipment, fresh solutions and gloves should always be used.

Potential Cause: Detection of the blocker by the primary/secondary antibodies:

  • Try adding a mild detergent (Tween 20) to the incubation and washing buffer. For phospho-specific antibodies, BSA should be used rather than milk. Milk contains casein which is a phospho-protein.

Potential Cause: Overexposure of Membrane

  • Reduce exposure times

Potential Cause: Membrane dried out

  • Make sure that membranes are adequately covered in buffer at all times.

Problem: Non-Specific Bands

Potential Cause: Antibody concentration is too high

  • Try decreasing the concentration of the primary antibody then try running a secondary antibody control.
  • Check the product datasheet/product page for recommendations on starting dilutions.

Potential Cause: Protein is overloaded

  • Try loading less protein into each well.

Potential Cause: Non-specific bands can be produced by unpurified antibodies

  • Switch to an affinity purified product instead

Potential Cause: Nonspecific sites were not blocked

  • Increase the concentration of the blocker from 5% to 7%
  • Increase the blocker incubation time
  • Increase the blocker incubation temperature
  • Try adding blocker to antibody dilution buffers

Problem: Band size is smaller than expected

Potential Cause: Protein sample is degraded

  • Make sure there is sufficient protease inhibitors in the sample buffer.
  • Make sure a fresh sample is used and kept on ice.

Problem: Band size is larger than expected

Potential Cause: Protein may form multimers

  • For extra time to break down the quaternary structure try boiling in Laemmli buffer.

Potential Cause: There are multiple modified forms of the protein sample (acetylation, methylation, phosphorylation etc.)

  • Try using a modifying agent to remove post-translational modifications, or try reviewing the literature for expected band size of modified forms.

Problem: Smile Effect

Potential Cause: Low resistance cause migration of protein to happen too fast

  • Decrease the voltage while running your gel

Potential Cause: Migration was too hot

  • Run the gel immersed in ice-cold buffer, on ice, or in a cold space.

Problem: Band Ran Too Far, or Not Far Enough

Potential Cause: Proteins were not adequately separated

  • Try changing the percentage of the gel, either by decreasing it for larger proteins, or by increasing it for smaller proteins
  • Try changing the run time of the gel, either by decreasing it for smaller proteins, or by increasing it for larger proteins

Problem: Patchy Background or Black Dots

Potential Cause: Uneven transfer of proteins to membrane

  • Try rolling out any air bubbles between the gel and the membrane to make sure there is an even transfer of proteins.

Potential Cause: Secondary antibody aggregates

  • Try spinning down the secondary antibody or removing aggregates through filtration.

Potential Cause: Antibodies are binding to the blocking agent

  • Try filtering the blocker to remove aggregates and contamination.

Potential Cause: Uneven coverage of membrane during incubation

  • Use a shaker to make sure that the membrane is covered evenly with solution.

About StressMarq Biosciences

Established in 2007, StressMarq Biosciences Inc. is a supplier of life science products that operates out of Victoria, Canada with a small, but dedicated group of scientists. Headed by our CEO and President Dr. Ariel Louwrier, StressMarq provides the research community with high-quality reagents backed with rigorous quality control data, expert scientific support, and fast international delivery.

“Discovery through partnership, Excellence through quality”

With over 7,000 products, our growth can be attributed to the continual production of cutting edge research products. Our diverse portfolio of primary antibodies, antibody conjugates, proteins, immunoassay kits and small molecules bridges across the life sciences, including products for cancer research, cardiovascular disease, cell signaling and neuroscience. To aid research worldwide, StressMarq has an extensive network of international distributors that allow us to supply reagents to over 50 countries.

In the years to come, StressMarq will continue to aid life science research by providing “Discovery through partnership, and Excellence through quality”.


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Last updated: Jan 28, 2020 at 2:20 AM

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