Bio-Rad Clarity™ Western ECL Substrate User Manual
Page 14
10 Technical Support: 1-800-424-6723 • www.bio-rad.com
Troubleshooting, continued
Problem
Cause
Solution
Poor antibody
binding to the
antigen
•
Detergents may affect
the binding of some
antibodies. Eliminate or
reduce their amount from
the assay
•
Increase the antibody
incubation times
Insuffecient
reagent
volume
•
Use additional volumes of
blocking, antibody, and
wash solutions
The enzyme
conjugate was
inactive
•
Test the reagent for
activity
•
Horseradish peroxidase
is most active at optimal
pH. Ensure excess wash
buffer is removed from
the membrane before
application of substrate
•
Sodium azide is a potent
inhibitor of horseradish
peroxidase. Eliminate
from antibody stocks
Blank spots
in areas of
membrane
that should
have signal
The
membrane
was allowed
to dry during
handling
•
Ensure that no air
bubbles were present
during assembly of
transfer stack
•
Ensure that warm
membranes are not
allowed to dry after
transfer