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High-throughput assays for sirtuin enzymes: A microfluidic mobility shift assay and a bioluminescence assay by Yichin Liu; Raphaele Gerber; John Wu; Trace Tsuruda; John D. McCarter is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.
What is High-throughput assays for sirtuin enzymes: A microfluidic mobility shift assay and a bioluminescence assay about?
Silent information regulator or sirtuin (SIRT) enzymes are b-nicotinamide adenine dinucleotide (oxidized) (NAD + )-dependent class III histone deacetylases. In this paper, two distinct assays to measure SIRT1 activity are described: a microfluidic mobility shift assay utilizing a fluorophore-labeled peptide substrate and a bioluminescence assay based upon quantitation of remaining NAD + . The mobility shift assay involves the electrophoretic separation of an N-acetyl-lysine-containing peptide substrate from deacetylated product which bears an additional positive charge. Interference from fluorescent compounds is minimized during screening by direct visualization of separated fluorophore-labeled substrate and product. A preferred peptide substrate for SIRT1 was identified using this assay. The NAD + bioluminescence assay couples NAD + consumption to the bacterial luciferase-catalyzed oxidation of decanal. This assay does not require synthesis of a labeled peptide and is applicable to sirtuins of any specificity with respect to peptide substrate. The stoichiometry between NAD + consumption and peptide deacetylation was shown to be 1:1 by the NAD + bioluminescence assay. Kinetic param
Who reads High-throughput assays for sirtuin enzymes: A microfluidic mobility shift assay and a bioluminescence assay?
It is typically read by researchers, students, and practitioners in Biochemistry, Genetics and Molecular Biology.
- Author
- Yichin Liu; Raphaele Gerber; John Wu; Trace Tsuruda; John D. McCarter
- Publisher
- Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)
- Published
- 2008
- Language
- EN
- Field
- Biochemistry, Genetics and Molecular Biology (Life Sciences)
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