Hi Suman

I have been using the Direct cAMP EIA kit from Enzo Life Sciences for a number of years and am very happy with the reliability of the kit.

Helen Steel

I believe that the Promega assay is fine if you are measuring relatively high levels of cAMP. We have used the Elisa kit from Assay Designs. Before that, we used 125I-based RIA kits, but people in the lab preferred to move away from using radioactivity.

Thank you Helen and Charles. I'll check those both out.

We have used the Cayman EIA kit for many years.

I have used Sigma direct cAMP ELISA kits and although very reliable they are very expensive. I now regularly use the Promega Glosensor reporter and in live cells, although I agree it may not be great for very small changes. I have compared forskolin concentrations with both ELISA results and luminescence produced in live Hek293 cells and found luminescence and cAMP concentration to have a first order polynomial relationship; therefore cAMP concentration can be estimated from luminescence. This would need to be repeated in your own hands with whatever cell/tissue/model you are using. But in general I love the Glosensor as it is cheap once up and running (if you have a luminescent 96 well plate reader) and you can get temporal readouts in live cells.

AlphaScreen® cAMP Assay Kit, 1,000 assay points by perkins elmer. The

detection of cAMP with AlphaScreen is based on the competition between cAMP produced by cells and a biotinylated cAMP probe that is recognized by the streptavidin-Donor and anti-cAMP conjugated Acceptor beads. The beads are brought into proximity and a signal is detected. Increased intracellular concentrations of cAMP following Gs coupled GPCR activation by an agonist results in the displacement of the biotinylated cAMP probe and leads to a proportional signal decrease. The effect of antagonists and reverse agonists can similarly be detected. We have used in a rat beta cell line..its also cheaper than anything else $300/1000 points but you need a flourescence plate reader

PDE activity is measured using the Phosphodiesterase [3H]cAMP Scintillation Proximity Assay (SPA) enzyme assay (Cat. #TRKQ7090, Perkin Elmer, Waltham, MA) following manufacturer’s protocol with minor modifications. The method in use has been reported in published literature with minor modifications (Chappie et al. 2007; Fawcett et al. 2000; Fujishige et al. 1999; Loughney et al. 1999; Soderling et al. 1999)

I have been using the DiscoverX cAMP assay kit. It's a competitive immunoassay, which directly measures the amount of cAMP produced by cells/tissue. The assay is simple, fast and reliable. You can find more details on this website http://www.discoverx.com/gpcrs/hithunter_camp.php

We've been using cAMP Kits from CisBio quite a bit in HT (compound) S and were pretty happy with it's reliability. They offer two types of kits, one for a great dynamic range and one for maximal sensitivity. It relies on direct competition of free cyclic cAMP with the labelled bound one. The variability is very low, also because of it's ratiometric nature. At the time we compared a few technologies. The cAMP Glo came out later. Sorry. Nevertheless the paper might be of some value to you..

High Throughput Screening Technologies for Direct Cyclic AMP Measurement. Daniela Gabriel, Magali Vernier, Martin J. Pfeifer, Boris Dasen, Laurent Tenaillon, and Rochdi Bouhelal. ASSAY and Drug Development Technologies. April 2003, 1(2): 291-303. doi:10.1089/15406580360545107.

But also be aware, that cAMP is a very sensitive messenger for general cell stress. Make sure you are not stressing your cells with your handling/pipetting. When I compared different cell dispensers using HTRF (CisBio) based cAMP detection I found that the additional levels of cAMP caused by the dispensing were marginal in comparison to the level caused by the fact that they were previously harvested and kept in suspension. Make sure your cells are unstressed when you stimulate them. Much of your variation can be caused by the way you handle your cells + liquid handling precision. For direct competition assays, you can actually adapt the assays's sensitivity and variation by fine tuning the amount of labelled cAMP. That gives you more precision and freedom than you will ever get with any ready made 'mix and measure' kit.

Yes, I have studied cAMP assays and also developed high-throughput screening. Amersham, Assay Design, Millipore, Luciferase, and CNG (cyclicnucleotide gated ion channels). I found Millipore is a simple, cheap and short time assay and working wonderful. Luci takes more time and require stronger hits and is not a robust for high-throughput screening. CNG assay is good for high-throughput screening. Depending on number of samples you can design which one would be suitable for your experiments. Thanks, and goodluck.

Syed

Hi, I have used Cayman kit  recently, and it worked well. Best, Eunus

 https://www.caymanchem.com/app/template/Product.vm/catalog/581001

Over-expressed cells with your target receptors containing Luciferse construct may work well with BrightGlo reagents but will take 4-6 hrs of stimulations in a high throughput  assay. It is highly sensitive depending on cell numbers per well. It requires optimization of cell numbers and dose-dependent stimulations of various compounds and ligands. Always you have to run in triplicate, however, the readout is simple and easy to analyze. This is suitable for drug discovery. 

I have successfully used the Cayman kit on both isolated cells and tissues over the years.

Dear all,

I would also like to buy a cAMP measurement kit and I would prefer to use one that relies on colorimetric measurement. That´s why I would like to buy either from Abcam (ab65355), Merck (116811) or Enzo (ADI-900-066). According to the information they provide, Abcam would be more sensitive than Merck...

Do you have any experience with Abcam or Enzo? I think Abcam and Enzo have been more used than the one from Merck but here you recommend the one from Merck.

Thank you!

We have actually started working with our mass spec facility to directly measure cAMP and cGMP levels. The reproducibility has been exceptional and since we are an internal user, it has been a free service!

I am working on patient sample so it is not possible for me to measure cAMP levels in fresh sample. Thereby, is there any possibility of measuring cAMP levels in stored samples?

Dear Syed A Morshed,

Could you please share the protocol for the cAMP Direct Immunoassay (Millipore)/ I mean the kit protocol, pdf?

Thanks