Western Blot FAQ

Troubleshooting Guide

Difficulties with Western blot assays can generally be broken down into three categories:

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1. No Signal or Weak Signal
 

Possible Cause

Solution
Cell or tissue type does not express the protein of interest
  • Perform a positive control
  • Preferably from cell or tissue lysate already verified to express the target protein
Improper cell treatment
  • Stimulate cells with the appropriate chemical, protein, etc.
  • Verify that the stimulation works
Improper sample preparation for gel loading
  • Ensure that the protein in the lysate is stable.
  • Use appropriate protease and phosphatase inhibitors, etc
  • Ensure protein samples contain SDS, and have been heated prior to gel loading
  • Include a reducing agent such as dithiothreitol (DTT) and/or 2-mercaptoethanol
Specific antigen concentration is too low
  • Load more protein on gel
  • Enrich the antigen by fractionation or by immunoprecipitation
Proteins did not transfer properly to the membrane
  • Wet PVDF membrane in methanol or nitrocellulose membrane in transfer buffer before use
  • Ensure there is good contact between the membrane and the gel
  • Optimize the transfer time
  • After transfer, ensure molecular weight markers were transferred
  • Stain the membrane with Ponceau red, and the gel with Coomassie blue
Insufficient antigen binding to membrane
  • If the antigen has low molecular mass, it may pass through the membrane
  • Switch to a membrane with a smaller pore size
  • Switch to a different type of membrane
The antigen is masked by the blocking buffer
  • Test different blocking buffers
  • Try milk, serum, BSA in Tris-buffered saline & PBS
  • Test different concentrations of each
Insufficient amount of antibodies present
  • Increase concentration of primary and/or secondary antibody
Antibody exposure time is too short
  • Increase the exposure time
Antibodies may have lost activity
  • Test antibodies by performing a Dot Blot
Excessive washing of the membrane
  • Reduce the number of washes
Substrate incubation is too short
  • Increase substrate incubation time
Substrate has lost activity
  • Test substrate using a positive control
Na azide inhibits enzyme reaction of HRP-conjugated Ab
  • Do not use sodium azide together with HRP-conjugated antibodies
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2. Nonspecific Bands
 

Possible Cause

Solution
Non-specific antibody binding
  • Reduce primary antibody concentration
  • Decrease the amount of total protein loaded on gel
  • Adjust membrane blocking conditions
  • Increase number of washes
  • Verify the specificity of the antibody
  • Blot with the secondary antibody alone.
  • If bands develop, choose an alternate secondary antibody
Degradation of protein
  • Prepare fresh samples
  • Use protease inhibitors during sample preparation
  • Minimize freeze/thaw cycles of sample
Aggregation of analyte
  • Increase the amount of DTT (20 -100mM) to ensure complete reduction of disulfide bonds
  • Heat in boiling water bath for 5-10 minutes before loading onto gel
Cell lines have been passaged extensively
Differences in protein expression profiles result
  • Go back to the original non-passaged cell line
  • Run the current and original cell line samples side-by-side
Protein has multiple modifications in vivo
Acetylation, methylation, glycosylation, phosphorylation, etc
  • Review the literature for modified protein variants
  • Adjust sample preparation accordingly
Target protein has multiple isoforms
Other proteins share similar epitopes
  • Check the literature for target protein isoforms
  • Perform a BLAST search to check for possible cross-reactions
  • Include other cell or tissue types
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3. High Background
 

Possible Cause

Solution
Too much protein per lane
  • Titrate down the amount of protein loaded per lane
Insufficient blocking of non-specific binding
  • Adjust blocking conditions
  • Include blocking agent in the antibody buffers as well
The primary antibody concentration may be too high
  • Titrate the antibody to find the optimal concentration
The secondary antibody may be binding non-specifically
  • Blot with the secondary antibody alone
  • If bands develop, choose an alternate secondary antibody
Incubation temperature may be too high
  • Incubate blot at 4°C
Cross-Rxn between blocking agent & primary or secondary Ab
Antibody cross-reacts with casein, a milk phosphoprotein
  • Add a mild detergent, e.g.Tween® 20, to incubation & washing buffers
  • Recommended for phosphoprotein specific antibodies
  • Use BSA as a blocking reagent instead of milk
Washing of unbound antibodies may be insufficient
  • Increase the number of washes
The membrane may give high background
  • Nitrocellulose membrane may give less background than PVDF
The membrane has dried out
  • Avoid drying out the membrane during processing and incubation
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