Western Blot Troubleshooting Guide

Western Blot is one of the routine methods in today’s cellular- and molecular biology labs. However, analyzing a western blot result can cause problems for users, since obtained data might be inconclusive. To help you with your next assay we put common problems and solutions together in one guide.

One of the reasons might be the wrong choice of antibodies. Try our webshop to find the right primary antibody and secondary antibody, simply choose “Western Blot” in the application filter. In case you have any further questions, just ask one of our experts.

Read this troubleshooting guide and become a western blot expert:

No Signal/ Low Sensitivity

Problem
Cause / Remedy

Analyte not detected.

  • Check the amount of protein used:
  • Bradford-Test
  • BCA-Method
  • Increase the concentration of protein
  • If possible, use a positive control with a high antigen expression level
  • Use a fresh lysis buffer
  • Add a protease Inhibitor to your sample buffer
  • Store samples on ice (4°C)

Analyte runs out of gel during electrophoresis

  • Use the protein ladder to ensure that the protein of interest (kDa) is still within the gel.
  • Shorten the time of electrophoresis

No transfer from gel to membran

  • Check if protein transfer has been successful:
  • Ponceau S Staining
  • Use a loading control (Housekeeping) to ensure that proteins have been transferred equally.
  • Increase duration of transfer

Wrong blotting membrane

PVDF

  • Need to be activated with methanol
  • High binding capacity (170 bis 200 μg/cm2)
  • More background compared to nitrocellulose membranes
  • Suitable for stripping-buffers and reprobing.

Nitrocelluse

  • Less binding capacity (80 bis 100 μg/cm2)
  • Less background

Choose the right pore size for your sample:

  • 0.1, 0.2 oder 0.45μm. (0,45μm pore size is suitable for most proteins (≥ 20kDa))

Transfer condition

  • Remove any air bubbles between gel and blotting membrane before transfer.
  • Reduce duration of transfer and/or current („Over transfer“).

Duration of blocking

Reduce duration of blocking

  • 1h at room temperature

Choice of blocking reagent

Antigen-Mask

  • Use a lower concentration
  • Use a different buffer

We recommend to use 5% normal serum from the same species as the secondary antibody or IgG-free BSA.

Strong washing

  • Reduce the number and/or duration of washing steps
  • 3x 5 Min (RT)
  • Reduce the concentration of detergent
  • 0.1-0.5% Tween 20

Wrong primary antibody

Ensure that your antibody of choice is suitable for western blot (datasheet)

Incubation time (primary antibody)

Expand duration of incubation

  • 4°C over night

Low concentratioon of antibody

  • Check antibody concentration used.
  • Validate affinity
  • Start with a higher concentration as stated in the datasheet. Then decrease concentration step by step.

Wrong secondary antibody

Check if secondary antibody bind to the primary antibody of choice (datasheet)

Horseradish peroxidase (HRP) activity inhibited

  • Do not use sodium azide in your buffer. As it inhibits the HRP (Horseradish peroxidase)-conjugate activity.
  • Do not use Glycerol with ACS grad below 99,5%. As it inhibits the HRP (Horseradish peroxidase)-conjugate activity.
  • Avoid repeated freezing and thawing of antibody solutions.
  • Prepare small aliquots

Storage Tips for Antibodies

ECL reagent expired

Use a fresh ECL reagent

ECL reagent contaminated

Use a fresh ECL reagent

Exposure time

  • Depending on the antigen expression level time of exposure may vary
  • Increase time of exposure by small steps

High Background

Problem
Cause/ Remedy

High antigen expression level

Reduce the concentration of protein

Choice of blocking reagent

  • Do not use Milk powder in case phosphorylated proteins need to be detected.
  • Primary antibody might cross-react with casein
  • Do not use Milk powder in case streptavidin/avidin conjugates are used.
  • Milk contains endogenes biotin
  • BSA and Milk powder are not suitable for secondary antibodies that detect goat, bovine or sheep.
  • Secondary antibodies might cross-react with bovine Immunoglobulines from BSA or milk powder

We recommend to use 5% normal serum from the same species as the secondary antibody or IgG-free BSA.

Incubation time (Blocking reagent)

Increase duration of incubation

  • 1h at room temperature

Wrong concentration of primary antibody

  • Reduce the concentration of the primary antibody
  • Titration

Wrong concentration of secondary antibody

  • Reduce the concentraion of the secondary antibody
  • Titration

Washing

  • Increase the number and/or duration of washing steps
  • 3 x 5min – 5 x 5min
  • Add a Tween 20 to your washing buffer
  • (0.1-0.5%)
  • Use a shaker

Exposure time

Depending on the antigen expression level time of exposure may vary

Transfer membrane

  • Use forceps when handling the membrane
  • Membrane should not dry out

Detection reagent

Reduce the concentration of substrate

Unspecific bands or dots

Problem
Cause/ Remedy

Protein is degraded

  • Use a fresh lysat
  • Store samples on ice (4°C)
  • Add a protease inhibitor to your sample buffer

Sample condition

  • Wrong isoform is detected
  • Posttranslational modification
  • Changes in molecular weight, might cause double bands
  • If possible, use a positive control
  • If possible, use a negative control
  • Bacterial contamination of sample and/or buffer
  • Prepare a fresh sample or buffer
  • Use deionized water

Transfer membrane

  • Remove any air bubbles between gel and blotting membrane before transfer.
  • Give your membrane a quick wash before blocking to remove any remaining pieces of gel or SDS.

Primary antibody

  • Verify specific binding of primary antibody
  • Use secondary antibody without primary antibody in a control experiment.
  • Optimize antibody concentration (titration).