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...
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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
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