Western Blotting Troubleshooting: Unusual or Unexpected Bands

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 concentrations