Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 3)

Section 1
Preparation
1.1 Introduction
Opti-4CN is an improved and more sensitive version
of the colorimetric horseradish peroxidase (HRP) sub-
strate, 4-chloro-1-naphthol (4CN). Opti-4CN may be used
simply as a replacement for 4CN resulting in a 4–8 fold
increase in detection sensitivity. When used in conjunction
with the signal amplification reagents in the Amplified
Opti-4CN substrate and detection kits, sensitivity may be
improved another 4–8 fold, resulting in an overall improve-
ment of 16–64 fold. The kits provide reagents for ampli-
fication and/or detection on 2,500 cm
2 of membrane.
1.2 Method Overview
The first step in western blotting is the transfer of anti-
gen onto a solid support membrane by one of several meth-
ods. The transfer can be done electrophoretically, following
separation of the antigen in a polyacrylamide or agarose
gel, passively by directly spotting the antigen onto a mem-
brane, or by vacuum filtration using a microfiltration appa-
ratus. Following antigen binding, the remaining protein
binding sites on the membrane surface are blocked to min-
imize non-specific interactions.
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