Assay Report Card

ID: ALA3888542

Type: Binding

Description: Inhibition Assay: Different concentrations of TA, TB, TH, or TM were pre-incubated with 1 μM of the sirtuin and 1 mM NAD in 20 mM Tris-HCl buffer (pH 8.0) with 1 mM dithiothreitol (DTT) at 37° C. for 30 minutes. Then 0.05 mM acyl peptides (H3K9Ac for Sirt1, Sirt2 and Sirt3; H3K9Su for Sirt5; H3K9Myr for Sirt6 and Sirt7) were added to initiate the reactions. The total reaction volume iwas 60 mL. Then reactions were incubated at 37° C. for a certain amount of time (2 minutes for Sirt1, 10 minutes for Sirt2 and Sirt5, 30 minutes for Sirt3, and 1 hour for Sirt6 and Sirt7). The reactions were stopped by adding 60 mL of an aqueous solution containing 200 mM HCl and 320 mM acetic acid. After centrifugation to remove precipitated proteins, the supernatant was analyzed by HPLC with a reverse phase C18 column (250×4.6 mm, 90 A, 10 mm, GraceVydac, Southborough, Mass.), with a linear gradient of 0% to 20% B for 10 minutes (1 mL/min).