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Western Blot Troubleshooting

Related Links

Western blot handbook

Troubleshooting guide

Recommended loading controls

Sample preparation

Running the SDS-Page gel

Transferring protein from gel to membrane

Immunoblotting and detection

Membrane stripping and reprobing

Blot storage

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The following troubleshooting guide is intended to explain causes and possible solutions for common problems observed in western blotting.

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No Signal or Weak Signal

Primary antibody concentration is too low

  • Increase the concentration of the primary antibody (titrations may be helpful). Novus Antibody Concentration Kit can be used to increase primary antibody concentration.
  • Increase the incubation time to 4°C overnight
  • If the primary antibody is re-used too many times, the effective antibody concentration may be too low; use fresh antibody to improve signal

Target protein concentration is too low

  • Load more protein per well (titrations might be helpful)
  • Use a positive control lysate known to express the target protein, an overexpression lysate, or a recombinant protein
  • Ensure the lysis buffer is the optimal buffer for the target protein; lysis buffer differs based on target protein localization.
  • Use immunoprecipitation or fractionation (i.e. nuclear fractionation) if necessary to increase the concentration of a non-abundant protein
  • Include protease inhibitors in the lysis buffer
  • Ensure the sample has not degraded

Protein transfer from gel to membrane was unsuccessful

  • Confirm the proteins were successfully transferred to the membrane by Ponceau staining of the membrane or Coomassie staining of the gel.
  • Confirm equal transfer by analyzing loading control expression

Primary and secondary antibody are not compatible

  • Ensure the secondary antibody was raised against the species in which the primary was raised
  • (e.g. if primary was raised in mouse, then use an anti-mouse secondary antibody)

Membrane choice was not ideal

  • Check the hydrophobicity/hydrophilicity of the antigen sequence. PVDF membrane may work better for hydrophilic/polar/charged antigens, while nitrocellulose may work better for hydrophobic/non-polar antigens

There are issues with blocking

  • Blocking for too long can mask certain epitopes and inhibit antibody binding; reduce the blocking incubation time or the concentration of blocking solution
  • Switch to a different blocking solution

Excessive washing of membrane

  • Detection reagents can become inactive over time. Ensure the reagents are fresh. The secondary antibody can be tested by dotting it on the membrane and incubating the blot with detection reagent.
  • Use more sensitive reagents when working with low abundance proteins (titrations may be helpful; if diluting, use high-purity water)

Image exposure was too short

  • Increase exposure time (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. Wet transfer is recommended for small protein, as well as membranes with smaller pore sizes (0.2 μm vs. 0.45 μm)

Sodium azide contamination has occurred

  • Sodium azide (often used to store primary antibodies) inhibits HRP activity. Ensure sufficient washing to remove the presence of sodium azide or use sodium azide-free buffers.

High Uniform Background

Insufficient blocking

  • Increase blocking time and/or temperature
  • Increase the concentration of blocking reagent (try up to 10%)
  • Consider changing the blocking agent (milk vs. BSA)
  • Include the optional blocking agents in antibody buffers (can also increase %)

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 primary or secondary (titrations may be helpful)
  • Include blocking agent in antibody buffers
  • Confirm the secondary antibody is specific by performing a secondary antibody control: Omit the primary antibody and only incubate the blot with the secondary antibody.

Insufficient washing of unbound antibodies

  • Increase the number and/or time of washes

Dry membrane

  • Make sure the membrane never becomes dry during the western blotting protocol

Film exposure is too long

  • Lower the exposure time (may be necessary to test a range of exposure times)

Detection reagents are too sensitive

  • Dilute the detection reagent in pure water or use a less sensitive detection reagent

Non-specific bands/wrong size or multiple bands

Target protein is less abundant than the threshold of non-specific binding

  • Load more protein in the SDS-PAGE gel
  • Enrich low-abundance proteins by immunoprecipitation or fractionation

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 (cleavage from a pro-form to a mature form). To confirm specificity, perform positive and negative controls such as recombinant protein or overexpression lysate, downregulated knockdown/knockout lysate

Speckled or Swirled Background

Membrane mishandling

  • Minimize contact with membrane. 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 the secondary antibody using a 0.2 μm filter to remove aggregate

Insufficient washing

  • Increase the volume of the washing buffer
  • Increase the number and/or duration of the washes

Other Issues

White/hollow bands

  • ECL substrate was consumed too fast. Decrease the concentration of primary/secondary antibody or use less protein

Smeared bands/lanes (sample overloading)

  • Load less protein into each lane

“Smiling” bands

  • Migration was too fast; decrease the voltage while running the gel
  • Migration was too hot; run the gel in the cold room

Molecular weight marker lane is black

  • The antibody may react with the molecular weight marker
  • Add a blank lane between the molecular weight marker and the first sample lane

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