(Re-sending, because this does not appear to have been circulated. VN)
In our lab Ritwick Sawarkar measured cAMP levels in developing cells of D.
discoideum using a standard radio-immunoassay with I125-labeled cAMP (made
by iodination of cAMP) and a home-made antibody. Similar antibodies are
commercially available. The estimates of cAMP levels seemed reasonable.
Interestingly, following starvation he could detect robust oscillations
with the expected period. See Sawarkar et al., "Histone Deacetylases
Regulate Multicellular Development in the Social Amoeba Dictyostelium
discoideum", J. Mol. Biol. 391:833–848, 2009.
Vidyanand Nanjundiah
Indian Institute of Science
Bangalore
---------------------------- Original Message ----------------------------
Subject: Re: [DICTY - 672] how to measure cAMP?
From: "Miranda, Edward Roshan" <[log in to unmask]>
Date: Thu, September 29, 2011 20:54
To: [log in to unmask]
--------------------------------------------------------------------------
One note that I forgot to add was that With most ELISA kits, antibody used
against cAMP also binds the 2-deoxy form of cAMP used to stimulate cAMP
production and DTT that we add into assay as an inhibitor of
phosphodiesterase quenches any fluorescence signal. So be careful to test
for these interferences if you decide to go with ELISA kits.
Roshan
Dr. Gad Shaulsky's lab
Baylor College of Medicine
Houston, TX-77054
-----Original Message-----
From: DICTY [mailto:[log in to unmask]] On Behalf Of
Miranda, Edward Roshan
Sent: Thursday, September 29, 2011 10:01 AM
To: [log in to unmask]
Subject: Re: [DICTY - 670] how to measure cAMP?
Dear all,
We have been using a protocol that was sent to me by Dr. Pauline Schaap a
while ago when I posted a similar question on this listserv. The protocol
works with PKAR isolated from beef skeletal muscle/heart as a binding
protein to measure cAMP. This is based on the same principles as the
Amersham kit as described by Robert in this chain of emails. I have
attached these protocols for the reference of the community. This assay
can be done as a 96 well setup or in individual tubes.
if you don't want to go through the pain of isolating PKAR from beef
muscle, you can also work with PKAR bought from Sigma Aldrich (cat#
P5511). I have compared the two proteins (one from Sigma and homemade beef
extract) and they both work with similar binding efficiency.
Also attached is a paper from Biotechniques describing in detail
extraction of beef PKAR. It is similar to Pauline's protocol and can be
used as reference if you are keen on certain details.
Best
Roshan Miranda
Dr. Gad Shaulsky's lab
Baylor College of Medicine
Houston, TX-77054
-----Original Message-----
From: DICTY [mailto:[log in to unmask]] On Behalf Of
Robert Insall
Sent: Thursday, September 29, 2011 8:57 AM
To: [log in to unmask]
Subject: Re: [DICTY - 669] how to measure cAMP?
PS there's a protocol in the Methods in Molecular Biology edited by Ludwig
Eichinger and Francisco Rivero...
--------------------------------------
Professor Robert Insall
CR-UK Beatson Institute for Cancer Research Switchback Road, Bearsden
Glasgow G61 1BD, UK
Tel. (0/44) 141 330 4005
Web http://www.beatson.gla.ac.uk/robert_insall
--------------------------------------
--
This message has been scanned for viruses and
dangerous content by MailScanner, and is
believed to be clean.
--
This message has been scanned for viruses and
dangerous content by MailScanner, and is
believed to be clean.
|
