Hello,
I address this question to all experimentalists who aim to establish a photolysis assay with a caged cAMP compound, but want to avoid the 8-Br-cAMP derivative. It should avoid a heavily molecular biology based approach, since I have very limited possibilities and capabilities for such a solution.
1a)
For a motility assay of Dictyostelium discoideum, we have successfully stimulated cells of Dictyostelium by using flow photolysis of caged cAMP compounds.
1b)
However, the substance is being discontinued.
Now, only BCMCM-caged-8-Br-cAMP and other caged 8-Br-cAMP substances are being offered.
2)
Since the latter (1b) have not worked as successfully as the former did, we are looking for alternative setups.
Therefore I wanted to know how others solved this problem. Did anyone find an alternative setup to work also?
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3) Details on my organism, Dictyostelium discoideum:
We assumed that the membran permeability of the brominated analogues would be a main problem. Also, membrane-bound PDE are assumed to be necessary for a working motility assay. However, these details should not be the point; the question could concern many experimentalists.
we were normally using DEACM or BCMACM caged cAMP variants depending on experimental preference on release efficiency or solubility for release experiments in sperm. are you looking for a compound or a instrumental setup?
Hello Shatanik,
this is a good question you asked (compound or whole instrumental setup?) We have all the components of a setup here - instead of the compound. So I think: finding a suitable compound would be the easiest, fastest solution. Establishing a completely new instrumental setup, however, could take some time...
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About the hints you gave on your compounds and your setup: I should point out our demands to a compound in our setup are.
DEACM/DMACM should be good cages/compounds, especially due to their high extinction coefficient and a good chemical quantum yield. BCMCM (sic!) has worked in the past. All of them should be highly soluble in water and less membrane permeable, which is essential for us.
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Last, what has not proven to work so far:
Yet, all of them are only available with 8-Br-cAMP, which has not proven to work as a first messenger for the Dictyostelium experiments we run/want to run again. We considered: Either membrane permeability, binding kinetics to the extracellular receptor, or both of them.
I see, could not you find a synthetic chemistry group to collaborate who can produce it for you. we for example make these in house.
Yes - we focused on the former Hagen Group (they characterized various coumarin-based cages), who synthetized it in the past at FMP, Berlin. But now, the group does not exist anymore; Mr Hagen has retired.
You can contact [email protected]. He may help you with the compounds.
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