Source of Problem | Possible Cause | Action/ Solution |
---|---|---|
Antibody related
|
Concentration of primary and/or secondary antibody too high
|
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Primary and/or secondary binds to the blocking agent
|
|
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Antibody has reduced activity due to long term or improper storage
|
|
|
Phospho-specific antibody has reacted with casein (a phosphoprotein) in nonfat milk blocking agent
|
|
|
Secondary antibody is binding to blocking agent
|
|
|
Antigen related |
Non-specific interaction with genomic DNA in samples
|
|
Technique related |
Membrane blocking is not sufficiant
|
|
Membrane has dried out |
|
|
PVDF membrane has higher background
|
|
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Inadequate washing between incubations
|
|
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Incubation temperature too high |
|
|
Film over-exposed or blot developed for too long
|
|
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Buffer related |
Insufficient concentration of detergent in the buffers
|
|
Transfer, incubation, or blocking solutions are contaminated
|
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Western Blot Troubleshoot: Faint Bands or Weak Signal | Western Blot Troubleshoot: Patchy or Uneven Spots on the Blot |