Bio-Rad Clarity™ Western ECL Substrate - Manual (Page 6)

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Background on Chemiluminescence
Chemiluminescence is a chemical reaction that produces light
and has become a common detection method for western
blotting because of its high sensitivity. With chemiluminescent
western blots, a secondary antibody is conjugated to the enzyme
horseradish peroxidase (HRP). Once the secondary reagent is
bound to the target protein on the membrane, the membrane
is incubated with a solution containing the chemiluminescent
substrate (Fig. 1). In the presence of peroxide, the HRP enzymes
catalyzes the oxidation of luminol, which then generates light (Fig.
2). An enhancer is included in the substrate solution to increase
the longevity and intensity of the emitted light. Depending on the
substrate and enhancer formulation, the half-life of the
light-generating reaction can range from a few minutes to over
an hour. This light resulting from this reaction can be detected
with either film or a digital imaging system. When combined with
a Bio-Rad digital imager such as the ChemiDoc
™ MP, the
Bio-Rad Clarity substrate produces clear digital images that can
be directly used for analysis or publication.
Antibody Incubations
A typical immunodetection experiment system utilizes two sets of
antibodies.
• The primary antibody, which is directed against the
target protein (Antigen)
• The secondary reagent, in this case an antibody that
recognizes and binds to the primary antibody; it is
conjugated to an enzyme such as HRP, which will
convert the substrate into light, which is then detected
by an imager of film