Summary: Strategy for stripping Western blots and reprobing with
ECL Western blotting
Enhanced chemiluminescence has become the most commonly
used method for detecting proteins after Western blotting.
One of the many advantages it has over colorimetric detection
methods is the ability to re-use blots after initial
immunodetection and thus obtain further information.
A previous application note (1), following the method of
Kaufman et al (2), outlined a successful protocol for stripping
and reprobing of Western blots. Data (not shown) indicates
that in excess of 50% of some target proteins can be lost
when performing experiments where PVDF and nitrocellulose
blots are stripped and reprobed. It is therefore important to
consider which antigen is present in least abundance and
probe for this first. The following article gives an example
where three antibodies have been detected, stripped and
redetected with ECLTM Western Blotting Detection Reagents.
A suggested strategy for stripping and re-detecting in four
different situations is also included.