Western Blot Troubleshooting: 8 Western Blot Protocol Issues Solved

Western Blot Troubleshooting: 8 Western Blot Protocol Issues Solved


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Most of us have had trouble getting a western blot protocol to work at some point. Don’t get discouraged.

Follow our western blot troubleshooting guide to quickly 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 try incubating longer
  • Always optimize both primary and secondary antibodies with every experiment

Potential Cause: Primary antibody does not recognize the antigen

  • Check data sheets and reference material to verify that the antibody does detect the antigen in the species

Potential Cause: Primary and secondary antibody mismatch

  • The secondary antibody should be raised against the host species of the primary antibody.  For example, if the primary was raised in mouse (ie. Mouse Anti-HSP70) use an anti-mouse secondary (ie. Goat Anti-Mouse)

Potential Cause: Storage issues

  • Freeze/thaw cycles are detrimental to the antibody structure and can cause degradation.  It is best to aliquot antibodies into one time use vials upon delivery
  • Antibody was not stored as recommended.  Unfortunately this might require a new vial to be used instead

Potential Cause: Antigen level is too low

  • Load sufficient protein onto the gel (~20 µg)
  • It also may be necessary to induce cells to produce more protein before harvest
  • Ensure protein is not degraded

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

  • Concentrate your protein lysates by isolating the cellular compartment containing your protein of interest (ie. mitochondria or cellular membrane)
  • Try using ECL western blot rather than the colorimetric western blot

Potential Cause: Epitope is masked by the blocker

  • Use a different blocker agent
  • Optimize the concentration of the blocker

Potential Cause: Protein did not transfer

  • Optimize transfer protocol for specific protein

Potential Cause: Secondary antibody tag not visible

  • Ensure that the secondary antibody tag has not been exposed to excessive light
  • Use a longer incubation time

Potential Cause: Excessive Washing

  • Decrease the number of washing steps

Problem: High Background Staining

Potential Cause: High Antibody Concentration

  • Reduce the concentration of primary and/or secondary antibody
  • Always optimize both primary and secondary antibodies with every experiment

Potential Cause: Insufficient Blocking

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

Potential Cause: Insufficient Washing

  • Increase the volume and number of washes
  • Add or increase concentration of detergent in wash buffer (ie. 0.05% Tween-20)

Potential Cause: Contaminated Solutions

  • Be aware of potential contaminants.  Always use clean equipment, fresh solutions and wear gloves

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

  • Add a mild detergent (Tween 20) to the incubation and washing buffer. For phospho-specific antibodies, always use BSA rather than milk. Milk contains casein which is a phospho-protein

Potential Cause: Overexposure of Membrane

  • Reduce exposure times

Potential Cause: Membrane dried out

  • Ensure membranes are thoroughly covered in buffer at all times

Problem: Non-Specific Bands

Potential Cause: Antibody concentration is too high

  • Decrease the concentration of the primary antibody, and run a secondary antibody control
  • Refer back to the product datasheet/product page for recommended starting dilutions

Potential Cause: Protein is overloaded

  • Load less protein into each well

Potential Cause: Unpurified antibodies can produce non-specific bands

  • Try switching 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
  • Add blocker to antibody dilution buffers

Problem: Band size is smaller than expected

Potential Cause: Protein sample is degraded

  • Ensure that there is sufficient protease inhibitors in the sample buffer
  • Use a fresh sample and keep on ice

Problem: Band size is larger than expected

Potential Cause: Protein may form multimers.

  • Try boiling in Laemmli buffer for extra time to break down the quaternary structure

Potential Cause: The protein sample has multiple modified forms such (acetylation, methylation, phosphorylation etc.)

  • Use a modifying agent to remove post-translational modifications, or review the literature for expected band size of modified forms

Problem: Smile effect

Potential Cause: Migration of protein was too fast due to low resistance

  • 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

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

Problem: Patchy background or Black Dots

Potential Cause: Uneven transfer of proteins to membrane

  • Roll out any air bubbles between the gel and the membrane to ensure even transfer of proteins

Potential Cause: Secondary antibody aggregates

  • Spin down the secondary antibody or remove aggregates through filtration

Potential Cause: Antibodies are binding to the blocking agent

  •  Filter the blocker to remove aggregates and contamination

Potential Cause: Uneven coverage of membrane during incubation

  • Use a shaker to ensure that the membrane is evenly covered with solution
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