Measurements of cAMP - anyone using the method of Thakker et al.? - Need some help with the detection step (Jan/10/2006 )

Dear all

I'm planning to perform measurements of cAMP as described by Thakker et al. in Methods Mol Med. 2003;84:29-37. The method seems relatively easy when all solutions etc. are prepared and it seems to be possible to analyse large number of samples in each experiment. But I'm not sure how to do the final steps where the samples are collected on filters and radioactivity is measured. What filters are being used and how is the harvesting performed?
Any hints are greatly appreciated

Sonja

Dear all

I'm planning to perform measurements of cAMP as described by Thakker et al. in Methods Mol Med. 2003;84:29-37. The method seems relatively easy when all solutions etc. are prepared and it seems to be possible to analyse large number of samples in each experiment. But I'm not sure how to do the final steps where the samples are collected on filters and radioactivity is measured. What filters are being used and how is the harvesting performed?
Any hints are greatly appreciated

Sonja

Hi again

I'll try to describe the method as I have understood it. A central part of this assay, is a renal cortex extracts, which contains a protein that binds cAMP. The assay is a competition assay where radioactively labelled cAMP and the cAMP in your sample compete for binding sites in the renal cortex extract. After mixing extract, a known amount of 3H-cAMP and your sample (or different concentrations of "cold" cAMP to make a standard curve), you separate bound cAMP from unbound. And finally the amount of bound cAMP is estimated by transferring the extract-bound cAMP to glass fibre filters, adding scintillation fluid and counting radioactivity. This was the short version... (and I may have missed some important steps...).
My questions regard practical things. For instance, the volumes after finishing the reactions are relatively large (several milliliters) and the reactions are performed in glass tubes. I wonder what kind of glass fiber filters to use for the harvesting of my samples? One option is to use circular filters about 2 cm in diameter and our old-fashioned "water-vacuum-based" harvester. However, this harvester takes only 12 filters at the time and the number of samples I need to analyse will be relatively large (the standard curve alone will be at least 20 samples). That will also give a significant time delay from the first to the last sample transferred to filter - is that a problem? (I guess questions like these could only be answered of someone with detailed knowledge of this method...). Is it possible to transfer about half the volume of each glass tube to 96-well plates that take large volumes (about 2 ml wells, if I remember correctly), then harvest as we usually do onto a large filter usually used for 3H-thymidine measurements (it is made of glass fibre) and then transfer the rest of the sample volumes to the same wells and harvest again onto the same filter (I will then cut the filter in small pieces and transfer to tubes and add scintillation fluid)?. Any comments?

Sonja



I do not have access to this paper. Would you please give more detail about the method?

From what you have describe, I think you may need to use a harvester to harvest your cell onto a filters paper and read it with a machine.

Sorry I am not very helpful.
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