I want to evaluate the amount of internal cAMP levels. Unfortunately, I suspended my cells of desire in RIPA buffer. Is it possibly, despite the harsh buffer, to determine cAMP levels in an appropriate way?
Thank you very much in advance for any little advices.
Kind regards
Dear Gerrit,
I'm sure you can find a method to measure cAMP in samples lysed with RIPA buffer. Yet, it is probably best to fist find the "kit" you want to use e.g. ELISA, and then treat the samples accordingly. We normally use a phosphodiesterase inhibitor before we lyse the cells as cAMP is quickly degraded.
Note that "intracellular" cAMP in your case is only ok if you have washed the cells before lysing them with RIPA, but I guess you have done so.
what kind of cells do you have? and in what amount? if the cAMP concentration should be higher than 50 nM, then you can flash freeze your sample, add 0.1 M HCl, and detect the cAMP with RP-HPLC.
Dear Gerrit,
the problem is that the antigen-antibody reaction of the cAMP-immunoassay has to cope with the constituents of this buffer. I have some doubts about this.
cAMP determination in cells is running in our lab via radioimmunoassay. You can come and visit us in Berlin-Buch. I will be able to explain our approach.
If you did as said, using RIPA should not be a problem. It depends on how you want to measure cAMP levels, by RIA, ELISA or HPLC/MS. In case of ELISA some kits advice for changing buffer, also cAMP precipitation is possible. You can find useful resources here: http://link.springer.com/search?query=+cAMP+extraction+&facet-content-type=%22Protocol%22
Good luck and best wishes,
Erik
cAMP in acidic solution is stable, thus after resuspend your cells in normal PB, immediately add the same volume(1v/1v) of 3.5% perchloric acid and vortex briefly and then store samples at -80C for assay later. For cAMP assay, thaw the samples, and neutrolise the samples by adding a quater volume of 50% saturated KHCO3 solution, and half volume of 150mM K-phosphorus buffer pH7.5, 4mM EDTA. Now the samples ready for assay, usually using radio-immunoassay.
Dear Gerrit,
I used a kit based on Elisa method to measure intracellular cAMP and I used samples obtained by lysis with RIPA buffer!
You can use your samples of course!
Marianna Ranieri
You want the assay as simple as possible. Add 1/10 volume of 1 N HCl to the samples to make sure cAMP is stable. Assuming you are using ELISA or RIA , prepare a calibration set of known cAMP standards in the same way. You should run this set plus an expected "low" and 'high" cAMP containing sample from your experiment with the assa protocol. If on the signal to concentration curve of known cAMP standards brace the low and high samples within the close to linear range of the calibration curve, go ahead and process the samples . If the assay looks saturated. dilute the samples further in 0.1 N HCl as appropriate. If the assay looks insensitive, you should try acetylation of the samples which will make the assay at least 10 -fold more sensitive.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques