The second messenger cyclic AMP (cAMP) is an important regulator of cell proliferation and differentiation because of its ability to act as a mediator of a variety of growth factors and hormones. Several hormones and neuropeptides have been known to activate the cAMP pathway through the enzyme adenlyl cyclase. Furthermore, cAMP is an important modulator of neuronal, cardiovascular and glandular physiology. The conventional method of measuring cAMP is by radioimmunoassay (RIA) - whereby the succinyl ester derivative of cAMP is radioiodinated. However, the iodination of this succinyl ester is not easy. It is difficult to get a good quality of radioiodiated cAMP because of both labeling and purification problems, which give very high backgrounds in the RIA. During my Ph.D research I wanted to assay cellular supernatants for their cAMP content. I tried standardizing the RIA but in the end gave up because the signal to noise ratio was just too low.
Then I switched over to the cAMP Assay Kit by R&D Systems. The biggest advantage of this kit is that it is non radioactive, which means the assay can be performed on your bench and all the hassles of dealing with a powerful tracer like radioactive iodine are over. In addition, the sensitivity and specificity of the assay kit is comparable to the radioimmunoassay. The kit provides an excellent set of standards, which allow you to quantitate the precise amount of cAMP in the sample. The information booklet accompanying the assay is highly detailed and it gives all protocols, troubleshooting tips, inter and intra assay precision values and different methods to calculate the amount of cAMP in the sample. If you want to measure acetylated cAMP in your samples, there are elaborate protocols, from how to acetylate the samples to how to interpret data, which are a huge asset. And because the kit is ELISA based, your experiment is over within 4-6 hours unlike the RIA, which is a much more laborious and time-consuming process.
The only disadvantage of the kit is that it is expensive. The kit includes one 96-well plate and after setting up the standards and controls (in duplicates or triplicates), there are not too many wells left to run a large number of test samples. Hence, if you have to analyze hundreds of samples, it could become extremely expensive to use this kit. However, the accuracy and convenience of the kit makes it a great asset for any laboratory that is performing signal transduction research.
Piyali Dasgupta, Ph.D.
Post-Doctoral Fellow
Moffit Research Center