OGA activity can determin using a fluorogenic substrate 4-methylumlliferyl-N-acetyl-β-d-glucosaminide (4MU-GlcNAc) (Sigma M9659). 4-Methylumlliferyl (4MU) has excitation and emission wavelengths of 360 and 450 nm, respectively. When conjugat to GlcNAc (4MU-GlcNAc), fluorescence is quench. After removal of GlcNAc by OGA, 4MU fluorescence can quantifi and the results can correct for the background emission from buffer components and uncleav 4MU-GlcNAc (Sigma M2133). To control for potential activity of lysosomal hexosaminidases, reactions using tissue homogenates or cell lysates should include a physiological excess of GalNAc, and parallel reactions for each.
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Sample using an exogenous GalNAc-substrate in place of the GlcNAc-substrate should includ on the same plate. Detection of O-GlcNAcylat Proteins OGT and OGA enzymatic activities are important. But, in many circumstances, their activities are simultaneously increas or decreas in response to biological stimuli. Therefore, the biological Ghana Email List consequences of their regulation have to further assess by determining both the overall protein O-GlcNAcylation and O-GlcNAcylation of specific proteins. We first discuss antibody-bas approaches that many laboratories, with the ability to do immunoprecipitation and western blots, can use. Then, we discuss a click chemistry-bas derivatization method which can complement antibody-bas approaches.
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The Promise of a Golden Era for Exploring the Frontiers of Aging, Metabolism and Rox Biology frontiersinJianhua Zhang* Department of Pathology, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, Unit States From ancient times to the modern day extending longevity or even finding the elixir for eternal life has en a motivating quest for many civilizations. There are no shortage of Hollywood films and TV series that feature long-liv creatures: some heroes and others villains. Many of the ancient Greeks have what we would regard as a normal lifespan (Montagu, 1994; Batrinos, 2008). For example, Socrates TH Lists fore his untimely demise was in his 70s. Physicians had en direct to concoct potions to extend the life of emperors and the wealthy.