Sirt1 fluorometric drug discovery kit

SirT1 activity was measured using a SirT1 Fluorometric Drug Discovery Kit (ENZO Life Sciences International, Inc. PA, USA) according to the manufacturer’s instructions. This assay uses a peptide containing human p53 amino acids 379–382 (Arg-His-Lys-Lys(Ac)) as a substrate. SirT1 activity is proportional to the amount of Lys-382 deacetylation. Fluorescence was assessed in a Molecular Devices M2 plate reader (Molecular Devices Corporation, Menlo Park, CA, USA) with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Changes in SirT1 activity in livers were calculated against the mean value of SirT1 activity in control liver and expressed as percent of control.

Cell lysates were obtained as described in the Western blot section above. An amount of 350 µg of extracted proteins was diluted in a final volume of 500 µL with M-PER lysis buffer and incubated with 2 µg of rabbit anti-SIRT1(H300) primary antibody (1:400; Santa Cruz Biotechnologies, Santa Cruz, CA, USA, Cat. No. sc-15404) for 24 h at 4 °C. Next, 20 µL of Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnologies, Santa Cruz, CA, USA, Sc-2002) were added, followed by incubation at 4 °C overnight. The mixture was centrifuged at 2500 rpm for 5 min at 4 °C. The supernatant was discarded, and the co-IP products were washed five times with PBS. After the final wash, the precipitates were resuspended in 30 μL of assay buffer from the SIRT1 activity assay kit. Enzyme activity was assayed with SIRT1 Fluorometric Drug Discovery Kit (Enzo Life Sciences Inc., Farmingdale, NY, USA) according to the manufacturer’s instructions.

Sirtuin 1 (SIRT1) deacetylase activity was evaluated in the extracts at the concentration shown in Table 1, according to the manual instruction of SIRT1 Fluorometric Drug Discovery Kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA). The measured fluorescence was directly proportional to deacetylation activity of the SIRT1 enzyme in the sample and was evaluated (excitation, 360 nm; emission, 460 nm) in the Infinite 200 PRO series plate reader (Tecan GmbH, Crailsheim, Germany).

Sirt1 activity was measured using the SIRT1 Fluorometric Drug Discovery Kit according to the manufacturer’s instructions (Enzo Life Sciences). Briefly, this assay uses a small lysine-acetylated peptide, corresponding to K382 of human p53, as a substrate. The lysine residue is deacetylated by SIRT1, and this process is dependent on the addition of exogenous NAD⁺. The assay was carried out at 37 °C using Greiner white, small volume 384-well plates. First, 4 µL of substrate Fluor de Lys (final concentration 25 μM) were mixed with 4 µL of extract previously diluted 1/100 in assay buffer and 2 µL of the enzyme were added. After an incubation of 15 min at 37 °C, 10 µL of Developer 1X solution (composed by buffer, developer 5X, and Nicotinamide 50 mM) was added and incubated for 45 min at 37 °C.
Afterwards, the fluorescence was measured on a Polar Star Omega (BMG Labtech) plate reader. The fluorescence generated is proportional to the quantity of deacetylated Lysine (i.e., corresponding to Lysine 382). All measurements were performed in triplicate and the final DMSO concentration is 0.1 %. SIRT1 inhibitors nicotinamide (2 mM), suramin (100 µM), and sirtinol (100 µM) were used to confirm the specificity of the reaction. Calculation of net fluorescence included the subtraction of a blank consisting of buffer containing no NAD+ and was expressed as a percentage of control.

SIRT1 deacetylase activity was measured using a SIRT1 Fluorometric Drug Discovery Kit (Enzo Life Sciences) according to manufacturer’s instruction.

SIRT1 activity was determined by using the SIRT1 Fluorometric Drug Discovery Kit (BML-AK555-0001, Enzo) according to the manufacturer's protocol [17 (link)]. Briefly, for cell-free measurement of the reaction between H₂S and recombinant SIRT1, 10 μL SIRT1 protein (0.25 U) and 5 μL NaHS (0, 12.5, 25, 50, and 100 μmol/L) were incubated with 5 μL substrate (0.25 mmol/L) and 5 μL NAD (0.25 mmol/L) plus 25 μL assay buffer. For tissue SIRT1 activity measurement, the reaction system contains 10 μL kidney tissue homogenate, 5 μL substrate (0.25 mmol/L), and 5 μL NAD (0.25 mmol/L) plus 30 μL assay buffer. Both reactions were carried out at 37°C for 40 min and stopped by addition of 1x Fluor de Lys® Developer II plus nicotinamide (50 μL per well) (every 1 mL stop solution contains 760 μL assay buffer, 40 μL 50 mmol/L nicotinamide, and 200 μL 5x Developer II). Fifteen mins later, fluorescence values were measured on a fluorometric reader (Synergy™ Mx, USA) with excitation at 360 nm and emission at 460 nm.

The SIRT1 deacetylase activity assay was performed as previously described [58 (link)]. SIRT1 activity was evaluated using a SIRT1 Fluorometric Drug Discovery Kit (Enzo Life Sciences, Farmingdale, NY, USA). Briefly, total protein (10 μg) was dissolved in the assay buffer and incubated with the acetylated substrate (Fluor de Lys-SIRT1 substrate, 25 μM) and NAD⁺ (100 μM) at 37 °C for 45 min. The concentration of deacetylated substrates was measured after the addition of the developer, and fluorescence was detected using a fluorescence plate reader (GENios, TECAN Instrument, Salzburg, Austria) with excitation and emission wavelengths of 360 nm and 460 nm, respectively.