Western Blot Troubleshooting: Unusual or Unexpected Bands


Western Blot Detection of IP Samples - Pocket Guide

Western Blot Detection of IP Samples - New Pocket Guide

Our new pocket guide contains a set of steps to help you with your experimental design.


Sign up to Our Emails

Sign-up to receive our eNewsletters />

	We are constantly expanding our range of antibodies and reagents, resources and online tools to help you choose the right antibody, design experiments and achieve more.
<a class=

Be the first to know when we launch new products and resources to help you achieve more in the lab.

Follow   Facebook twitter Linkedin You tube Pinterest
Difference seen Possible Cause Action/ Solution
Band(s) at lower molecular weight than expected
  • Target protein has been cleaved or digested
  • Splice variants exist
  • Another protein bearing the same/similar epitope has been detected by antibody
  • Use a fresh sample which has been kept on ice
  • Add fresh protease inhibitors to the lysis buffer
  • Try alternate antibody
Band(s) at slightly higher molecular weight than expected, and may be blurred
Protein may be glycosylated or otherwise modified at one or more amino acid residues
  • Use enzymes to remove suspected modification returning molecular weight closer to expected
  • Check amino acid sequence and literature
Band(s) at significantly higher molecular weight than expected
Dimers, multimers, or protein-protein interactions may be occuring because samples have not been fully reduced and denatured.
  • Add fresh DTT or bME to samples and reheat before repeating experiment
  • Prepare new samples with fresh loading buffer
Multiple bands at various molecular weights
Primary antibody concentration may be too high, or there
is a cross-reactivity with similar epitopes on other proteins
  • Use an affinity-purified primary antibody
  • Optimize primary antibody concentration
  • Try another antibody
  • Check antibody specificity with blocking peptide
Secondary antibody concentration is too high leading to non-specific binding
  • Decrease/optimize the concentration of the secondary antibody
  • Use an affinity-purified secondary antibody
  • Repeat immunodetection with secondary antibody alone to check for non-specific binding
Protein exists in several different isoforms
  • Check literature
Bands are blurry Gel was run at too high a voltage
  • Repeat gel at lower voltage
Incorrect running buffer composition
  • Prepare fresh running buffer
Trapped air bubble present during transfer
  • Carefully remove air bubbles between the gel and the membrane before protein transfer
Bands are smile shaped, not flat
Running conditions were too fast
so gel became over heated
  • Check and optimize gel electrophoresis conditions
  • Run gel at 4°C
White (negative) bands on the film when using ECL detection
Too much protein has been loaded
  • Load less sample
  • Repeat with dilution series of sample
Antibody concentration is too high
  • Reduce/optimize the antibody

Chapter 6: Western Blot Troubleshooting Western Blot Troubleshoot: No Bands