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

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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

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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 4^oC 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

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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

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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

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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

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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

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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

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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