Yes! That's the problem that we had!

Terri 

Sent from my iPhone

On Sep 29, 2011, at 1:30 PM, "Miranda, Edward Roshan" <[log in to unmask]> wrote:

> 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
> --------------------------------------