Western Blot Doctor™ — Protein Band Size and Pattern Problems



The Western Blot Doctor is a self-help guide that enables you to troubleshoot your western blotting problems. In this section, you can find solutions to issues related to protein band size and pattern problems.

Other sections in the Western Blot Doctor:

Problems and Solutions

Click on the thumbnail that is most representative of your own blot to discover the probable causes and find specific solutions to the problem.

Band(s) at significantly higher MW than expected

A blot with non-specific-bands – Western Blot Doctor

Multiple bands at various MWs


Problem: Band(s) at lower MW than expected

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Possible sources of unexpectedly low-MW bands include protein cleavage or degradation, splice variants, and nontarget proteins bearing similar epitopes.
Possible causes: Solutions:
Target protein has been cleaved or digested
  • Use a fresh sample that has been kept on ice
  • Add fresh protease inhibitors to the lysis buffer (e.g., EDTA or PMSF)
Splicing variant exists
  • Confirm whether a splice variant may exist for your protein
  • Try an alternate antibody
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Another protein bearing the same/similar epitope is detected by the antibody
  • Make sure you include a negative control for the expression of your protein
  • Try an alternate antibody
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Problem: Band(s) at slightly higher MW than expected or blurry

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Bands at MW slightly higher than expected and/or blurred may indicate protein modifications such as glycosylation.
Possible causes: Solutions:
Protein may be glycosylated or otherwise modified at one or more amino acid residues
  • Use enzymes that remove suspected modification to restore molecular weight closer to expected
  • Check amino acid sequence for known motifs for posttranslational modifications, and search literature for other evidence of modified forms

Problem: Band(s) at significantly higher MW than expected

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Sources of unexpectedly high-MW bands include protein-protein interactions and antibody cross-reactivity.
Possible causes: Solutions:
Dimers, multimers, or protein-protein interactions occurring because samples have not been fully reduced or denatured.
  • Add fresh DTT or 2-mercaptoethanol to samples and reheat before repeating experiment to remove disulfide bonds
  • Try stronger reducing agents e.g., tryibutylpohsphine or TCEP
  • Add a chaotropic denaturant (e.g., urea) to denature the protein
  • Prepare new samples with fresh loading buffer

Problem: Multiple bands at various MWs

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Multiple nonspecific bands on the blot may be due to antibodies of poor quality or at too high a concentration, insufficient blocking, or nonspecific binding due to the presence of SDS. A blot with non-specific-bands – Western Blot Doctor
Possible causes: Solutions:
Primary antibody concentration too high or cross-reactivity with similar epitopes on other proteins
Secondary antibody concentration too high, leading to nonspecific binding
  • Decrease or optimize the concentration of the secondary antibody, e.g., using a checkerboard screening protocol
  • Use an affinity-purified secondary antibody
  • Repeat immunodetection with secondary antibody alone to check for nonspecific binding
Protein exists in several different isoforms
  • Check research literature for existence of isoforms or variants
Primary or secondary antibody contaminated with nonspecific IgG or with IgG cross-reactive among species


  • Use purified IgG primary antibody fractions and affinity-purified blotting-grade cross-adsorbed secondary antibody
Monoclonal antibodies reacted nonspecifically with SDS-denatured proteins
  • Compare the binding of other monoclonal or polyclonal antibodies
  • Blot native proteins as a comparison, e.g., by blue native PAGE
Nonspecific interactions occurring due to ionic associations; for example, avidin, a glycosylated protein, may bind to more acidic proteins on blots
  • Increase washing stringency:
    • Increase the ionic strength of the incubation buffers
      1. Increase the salt concentration of your TBS-T
      2. Try PBS-T instead of TBS-T (do not do this if using phosphospecific antibodies)
    • Increase the duration of washes
    • Increase the number of washes
    • Perform washes at room temperature
  • Include progressively stronger detergents in the washes; for example, SDS is stronger than Nonidet P-40 (NP-40), which is stronger than Tween-20
  • Include Tween 20 in the antibody dilution buffers to reduce nonspecific binding
  • Increase the Tween-20 concentration to 0.01–0.5% (v/v)
Insufficient blocking of nonspecific sites
  • Increase the concentration of blocking reagent (e.g., BSA, nonfat dry milk, etc.) from 5% to 7% (w/v)
  • Consider blocking overnight at 4ºC or at least 1 hour at room temp (increase length of incubations if necessary)
  • If not already included, add up to 0.01–0.5% Tween-20 to blocking buffer
  • Prepare antibody dilutions to the same blocking buffer with same increased concentration of Tween-20
SDS caused nonspecific antibody binding to immobilized proteins
  • Be sure to equilibrate gel with transfer buffer before transfer
  • If the transfer buffer contains SDS, be sure to include a wash step before performing the first antibody incubation step; wash step can be performed with washing buffer
  • If washing does not resolve the problem, consider avoiding SDS during blotting procedure if possible

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