Western Blot Troubleshooting: Unusual or Unexpected Bands

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Unusual or unexpected bands

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    - Guide: Introduction to Western Blotting
      - Chapter 6: Western Blotting Troubleshooting
        
        Western Blotting
        
        Western Blot: No Bands
        
        Western Blot: Faint Bands or Weak Signal
        
        Western Blot: High Background Signal on the Blot
        
        Western Blot: Patchy or Uneven Spots on the Blot

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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
White (negative) bands on the film when using ECL detection	Antibody concentration is too high	Reduce/optimize the antibody concentrations

Chapter 6: Western Blot Troubleshooting

Western Blot Troubleshoot: No Bands