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Western Blot Troubleshooting

The following troubleshooting guide is summarized to explain causes and possible solutions for common problems observed in western blot (WB) assay. Though the tips provided here cover many different problems you may encounter in WB, we hope that you will find the information beneficial to you and useful as a reference guide. The intact Western Blot Protocol is available for you or view WB procedure step-by-step. For further assistance, please contact us.


No Signal or Weak Signal

Possible Cause Recommended Solution
Primary antibody concentration is too low
  • Increase the concentration of the primary antibody.
  • Increase the incubation time to 4°C overnight.
  • Use fresh antibody to improve signal.
Target protein concentration is too low
  • Load more protein.
  • Choose the appropriate lysis buffer.
  • Use immunoprecipitation (IP) if necessary to increase the concentration of a non-abundant protein.
  • Add protease inhibitors into the lysis buffer.
  • Make sure the sample has not degraded.
Protein transfer from gel to membrane was unsuccessful
  • Make sure the proteins were successfully transferred to the membrane.
  • Confirm equal transfer by analyzing loading control expression or positive control.
Primary and secondary antibody are not compatible Ensure the secondary antibody was raised against the species in which the primary was raised.
Membrane choice was not ideal PVDF membrane may work better for hydrophilic/polar/charged antigens, while NC membrane may work better for hydrophobic/non-polar antigens.
Blocking Blocking for too long can mask certain epitopes and inhibit antibody binding. Reduce the blocking incubation time or the concentration of blocking solution.
Excessive washing Reduce the washing times appropriately.
Image exposure was too short Check several times to achieve optimal exposure time.
Antibody only recognizes native proteins Do not use reduced, denatured proteins if working with an antibody that only recognizes native proteins.
Targets are low molecular weight Reduce transfer time to prevent over-transfer.
Sodium azide contamination Sodium azide inhibits HRP activity. Ensure sufficient washing to remove the presence of sodium azide or use sodium azide-free buffers.

High Uniform Background

Possible Cause Recommended Solution
Insufficient blocking
  • Increase blocking time and/or temperature.
  • Increase the concentration of blocking reagent.
  • Changing the blocking agent.
Blocking not compatible For detection of phosphorylated proteins, milk is not recommended (milk and casein are phospho-protein rich).
Non-specific binding due to high antibody concentration
  • Lower the concentration of the antibody.
  • Add blocking agent in antibody buffers.
  • Confirm the secondary antibody is specific by performing a secondary antibody control.
Insufficient washing of unbound antibodies Increase the number and/or time of washes.
Dry membrane Make sure the membrane never becomes dry.
Film exposure is too long Lower the exposure time.
Detection reagents are too sensitive Dilute the detection reagent or use a less sensitive detection reagent.

Non-specific Bands/Wrong Size or Multiple Bands

Possible Cause Recommended Solution
Target protein is less abundant than the threshold of non-specific binding
  • Load more protein in the SDS-PAGE gel.
  • Use immunoprecipitation (IP) if necessary to increase the concentration of a non-abundant protein.
Sample degradation
  • Use fresh lysates.
  • Keep sample on ice until just before sample buffer addition and boiling.
  • Always include protease inhibitors and phosphatase inhibitors if detecting phosphorylated target.
Other protein isoforms may be present Alternative splicing or multimer formation may be present. In this case, an isoform-specific antibody may be required.
Post-translational modifications may be present Predicted molecular weight can be influenced by many factors such as glycosylation, phosphorylation, and protein processing. To confirm specificity, perform positive and negative controls such as recombinant protein or overexpression lysate, downregulated knockdown/knockout lysate.

Speckled or Swirled Background

Possible Cause Recommended Solution
Membrane mishandling Use clean tools to handle the membrane.
Buffer contamination Make fresh buffers.
Air bubbles Roll out any bubbles between the gel and membrane before transfer.
HRP aggregation Filter to remove aggregate.
Insufficient washing
  • Increase the volume of the washing buffer.
  • Increase the number and/or time of the washes.

Uneven White Spots

Possible Cause Recommended Solution
Air bubbles were trapped against the membrane during transfer or the antibody is not evenly spread on the membrane. Make sure you remove bubbles when preparing the gel for transfer. Incubate the antibodies while agitating.

Uneven Staining of the Gel

Possible Cause Recommended Solution
Contamination from bacteria Keep antibodies at 4°C and use fresh buffers covering the gel.
Not enough antibody volume Make sure the membrane is covered with the antibody and incubate while agitating.

Smile Effect on the Bands

Possible Cause Recommended Solution
Migration was too fast Decrease the voltage while running the gel.
Migration was too hot Run the gel in the cold room or on ice.
For research use only. Not intended for any clinical use.