Difference seen | Possible Cause | Action/ Solution |
---|---|---|
Band(s) at lower molecular weight than expected
|
|
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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
|
|
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. |
|
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
|
|
Secondary antibody concentration is too high leading to non-specific binding
|
|
|
Protein exists in several different isoforms
|
|
|
Bands are blurry | Gel was run at too high a voltage |
|
Incorrect running buffer composition
|
|
|
Trapped air bubble present during transfer
|
|
|
Bands are smile shaped, not flat
|
Running conditions were too fast
so gel became over heated
|
|
White (negative) bands on the film when using ECL detection
|
Too much protein has been loaded
|
|
Antibody concentration is too high
|
|
Chapter 6: Western Blot Troubleshooting | Western Blot Troubleshoot: No Bands |