Trying to develop a functional assay for a GPCR. Initially had trouble getting data in functional assay with vs. without agonist, because commercial membrane preps didn't contain sufficient G-protein. Now we have sample from collaborator which should contain G protein and purified receptor. Only have limited supply to test. Need to determine if it makes sense to run functional assay with BODIPY-GTPyS with vs. without agonist.
Will BODIPY-GTPyS transport through lipid membrane? Will it get stuck in membrane? Any advice would be greatly appreciated, thanks!
Mike, the mebrane prep must contain membrane vesicles turned inside out, so GTPgammaS, or in your case its BODIPY-derivative shuld be able to react with G protein in the prep. It is very likely not transported through the membrane and it cannot cross the membrane, but this will not affect your measurement. After proper time allowed for the binding, you supposed to separate the unbound dye-GTP from the bound ones with filtration.
Moreover, I suggest you to do membrane prep by yourself. It takes not much more than two consecutive centrifugations...
Best, Karoly
- Badges
- Science topic
- Similar topics
- Geoscience
- Cartography
- Mapping