Bio-Rad Clarity™ Western ECL Substrate - Manual (Page 12)

8 Technical Support: 1-800-424-6723 • www.bio-rad.com
Troubleshooting
Problem Cause Solution
High
background
Blocking was
incomplete
• Increase the
concentration of blocking
agent or increase
blocking duration
• Match the blocker to
the membrane. For
example, gelatin may give
poor results on PVDF
membranes
Washing was
insufficient
• Increase the number,
duration, or stringency of
the washes
The primary
or secondary
antibody
was too
concentrated
• Decrease antibody
concentrations
• Perform a dot-blot
experiment to
optimize the working
concentrations
Areas of no
signal within
a band
(a donut
appearance)
Localized
substrate
depletion
• Bands on the blot with
high protein amounts
will lead to excessive
local concentrations of
peroxidase conjugate.
This may lead to localized
substrate depletion. Use
lower concentrations of
primary and secondary
antibody
• Load less sample