Bio-Rad Immun-Blot® AP Colorimetric Kits User Manual

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Section 1
Preparation

1.1 Introduction

The Immun-Blot assay kits are enzyme immunoassay kits

optimized for the detection of specific antigens, at picogram levels,
following electrophoretic blotting or dot blotting to a membrane. The
kits provide all necessary components and chemicals, in an easy-to-use
form, for detection of selected purified proteins using a double
antibody or antibody-binding protein blotting assay.

The Immun-Blot alkaline phosphatase and Immun-Blot horseradish

peroxidase assay systems can be used to detect rabbit, mouse, or human
immune complexes.

They will give sensible detection of specific

antigens or cloned translation products following dot-blotting,

electrophoretic blotting,

3-11

filter affinity transfers,

and in situ colony

or plaque lifts.

The Immun-Blot assay is fast and simple. Antigen is transferred

and bound to the membrane. This transfer can be done electro-
phoretically, following separation of the antigen in a polyacrylamide or
agarose gel, or passively by either directly spotting the antigen to a
membrane or by overlaying a phage plate or lysed bacterial colony with
nitrocellulose membrane to bind expressed proteins. Following binding
of antigen, the remaining protein binding sites on the membrane
surface are blocked with gelatin or equivalent proteins which will not
react with the primary and secondary antibodies. BSA (not included in
the kit) is used as a blocker when the Bio-Dot

or Bio-Dot SF

apparatus is used for blotting, since gelatin will obstruct filtration
through blotting membranes.

The membrane with bound antigen is then incubated with first

antibody, specific for the antigen to be detected. The membrane is

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