More questions on cAMP assays

[James J. Campanella]

Dear colleagues,

I posted a question several days ago on where I
might find a non-isotopic assay for cAMP levels.
I received several answers. Most of these answers
pointed me to an immunoassay kit from Amersham. 
The cAMP immunoassay kit from Amersham has only
one problem-- since it is performed on microtiter
strips, it requires a $3000 microtiter reader to
be of much use. Since I wanted to perform the
assay in connection with an undergraduate teaching
lab, buying a microtiter reader is a bit impractical. 

One person suggested that I buy the cAMP antibodies
from Sigma. I checked into this and found that
Sigma's price for these antibodies was quite
reasonable. This got me to thinking along the lines
of a putting together a "home-made" assay of sorts. 
The basic concept is outlined below. I wanted the
opinion of others on the idea. . . . 
-----------------------------------
To assay cAMP levels, we can perform a dot-blot immunoassay. 
We can blot bacterial extracts alongside controls of
known cAMP concentration on to nitrocellulose. The blot
can be crosslinked, blocked, and then treat with 
an anti-cAMP antibody (rabbit antibody, Sigma Cat#
A0670, $62.15). A labelled-secondary antibody can 
then be bound to the first (anti-rabbit Immunoglubulins 
conjugated to alkaline phosphatase, Sigma Cat#A2306, $49.95).
We can then wash off non-specific antibodies and visualize 
with a standard chromogenic substrate (e.g. BCIP, BMB Cat#
760994, 250 mg, $45.00 ) used in non-isotopic Southern blot
visualization. The students will Make a visual comparison
between standard and  experimental spots to determine cAMP 
concentrations.
-----------------------------------
The biggest problem that I see is whether or not 
the cAMP will actually bind and crosslink to the
nitrocellulose. If it doesn't bind, then the assay
can't be performed this way. Oh, and thanks to 
everybody who has already responded. I'm grateful.

Jim Campanella
Lehigh University
Dept. of Biological Sciences
Bethlehem, PA
-- 
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