Bio-Rad Clarity™ Western ECL Substrate - Manual (Page 13)

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Troubleshooting, continued
Problem Cause Solution
No reaction
or weak
signal
Proteins may
be washed
from the
membrane
during assays
• Reduce the number or
stringency of washes
Antigen
binding to the
membrane
was
insufficient
• Decrease antibody
concentrations
• Stain the gel after
transfer or use
prestained standards to
assess transfer efficiency
• Some total protein
stains (such as amido
black and colloidal gold)
interfere with antibody
recognition of the
antigen. Do not use a
total protein stain, or use
a different stain
• Optimize the blocking
reagent. Some blocking
reagents (such as
nonfat dry milk) provide
high stringency at the
expense of sensitivity.
Others (such as BSA)
offer comparatively high
sensitivity at the expense
of higher background or
nonspecific binding