Sirt1 assay kit

Sirt1 activity was measured using a SIRT1 assay kit (Sigma, USA) according to the manufacturer’s protocol. Briefly, 20 μl of protein was blended with 5 μl of NAD+ solution, and 10 μl of SIRT1 substrate solution was then added to the mixture, which was then incubated at room temperature for 10 min. Thereafter, 5 μl of developing solution was added, and the samples were incubated at 37°C for 10 min. Fluorescence was read using a plate reader, and the activity of the samples was calculated using a standard curve.

SIRT-1 activity was determined using the SIRT1 Assay Kit (Sigma Aldrich) following manufacturer instructions and fluorescent spectrometer (Biorad VersaFluor, Marnes-la-Coquette, France) set with 340 nm excitation and 430 nm emission wavelengths. The relative SIRT1 activity was revealed as a percentage relative to the corresponding control (adding same volume of extraction solvent) for every extract.

Total protein was extracted from the colon tissue and the level of SIRT1 activity was tested using a SIRT1 Assay Kit (#CS1040, Sigma-Aldrich, United States) according to the manufacturer’s instructions.

Whole cell lysates or tissue homogenates were prepared using the NETN buffer (20 mM Tris pH8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) with freshly added protease inhibitor tablet and SIRT1 activities were measured with a SIRT1 assay kit (Sigma) according to vender’s recommendations.

SIRT1 deacetylase activity was quantified using the fluorometric SIRT1 Assay kit (Sigma-Aldrich, St. Louis, USA) according to the manufacturer's instructions. Fluorescence intensity at 444 nm (excitation 355 nm) was recorded and normalized to micrograms of protein. Values are represented as -fold of NG control.

SIRT1 activity was measured by using the SIRT1 Assay Kit (Sigma). In this assay, SIRT1 activity is assessed by the degree of deacetylation of a substrate which represents a peptide containing amino acids 379-382 of human p53 (Arg-His-Lys-Lys[Ac]). SIRT1 activity is directly proportional to the degree of deacetylation of Lys-382. Nuclear cell lysates (quantity) were incubated with peptide substrate (25 μM) in a phosphate-buffered saline solution at 37°C for 45 minutes. The reaction was stopped with the addition of 2 mM nicotinamide and a developing solution that binds to the deacetylated lysine to form a fluorophore. Following 10 minutes incubation at 37°C, fluorescence was read in a plate-reading fluorometer at an excitation wavelength of 360 nm and an emission wavelength of 450 nm. In each assay, human recombinant SIRT1 enzyme (1 Unit per well), a SIRT1 activator, and suramin sodium (5 mM), a SIRT1 inhibitor were utilized as positive and negative controls in each set of reactions. A standard curve was constructed using deactylated substrate (0-10 μM). Data for endogenous SIRT1 activation were normalized to cellular protein concentration measured via BCA-assay.

SIRT1 deacetylase activity was measured using the fluorometric SIRT1 Assay Kit (CS1040; Sigma) following the manufacturer’s instructions. Briefly, the assays were performed by incubating different concentrations of PIC-OCT, including 1, 2, 4, 10, 20, and 40 µM, with SIRT1 substrate solution at 37 °C for 30 min. After the addition of the developing solution and incubation at 37 °C for 10 min, fluorescence intensity was measured at 460 nm (excitation 355 nm) using a Microplate Fluorescence Reader (Varioskan Flash, Thermo Fisher) and compared with a standard curve.