SIRT1 FRET-Based Screening Assay Kit

Cayman Chemical Item Number 10010991

SIRT1 FRET-Based Screening Assay Kit

Description

Sirtuins (SIRTs) represent a distinct class of trichostatin A-insensitive lysyl-deacetylases (class III HDACs). Human SIRT1 is the homolog of yeast Sir2 (Silent information regulator 2) and has been shown to regulate the activity of the p53 tumor suppressor and inhibit apoptosis. Small molecule activators of SIRT1, such as resveratrol, extend lifespan in yeast and C. elegans in a manner that resembles caloric restriction. Cayman’s SIRT1 FRET-based Screening Assay Kit provides a convenient fluorescence-based method for screening SIRT1 inhibitors or activators. The procedure requires only two easy steps, both performed in the same microplate. In the first step, the substrate, which is coupled to the fluorophore and quencher, is incubated with human recombinant SIRT1 along with its cosubstrate NAD⁺. Deacetylation sensitizes the substrate such that treatment with the developer in the second step results in the separation of the quencher and fluorophore. The resulting fluorescence can be easily analyzed using an excitation wavelength of 335-345 nm and emission wavelength of 440-465 nm.

Stability

1 year

Storage

-80°C

Shipping

Dry ice in continental US; may vary elsewhere

Background Reading

Grunstein, M. Histone acetylation in chromatin structure and transcription. Nature 389 349-352 (1997 Sep 25).

Imai, S., Armstrong, C.M., Kaeberlein, M., et al. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403 795-800 (2000).

Tanny, J.C., and Moazed, D. Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product. Proc Natl Acad Sci USA 98(2) 415-420 (2001).

Borra, M.T., Smith, B.C., and Denu, J.M. Mechanism of human SIRT1 activation by resveratrol. J Biol Chem 280(17) 17187-17195 (2005).

Wood, J.G., Rogina, B., Lavu, S., et al. Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature 430 686-689 (2004).

Howitz, K.T., Bitterman, K.J., Cohen, H.Y., et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425 191-196 (2003).

Saunders, L.R., and Verdin, E. Sirtuins: critical regulators at the crossroads between cancer and aging. Oncogene 26 5489-5504 (2007).

Longo, V.D., and Kennedy, B.K. Sirtuins in aging and age-related disease. Cell 126 257-268 (2006).

Langley, E., Pearson, M., Faretta, M., et al. Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence. EMBO J 21(10) 2383-2396 (2002).

Vaziri, H., Dessain, S.K., Eaton, E.N., et al. hSIR2SIRT1 functions as an NAD-dependent p53 deacetylase. Cell 107 149-159 (2001).

Luo, J., Nikolaev, A.Y., Imai, S., et al. Negative control of p53 by Sir2α promotes cell survival under stress. Cell 107 137-148 (2001).

Frye, R.A. Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. Biochem Biophys Res Commun 273 793-798 (2000).

Denu, J.M. The Sir2 family of protein deacetylases. Curr Opin Chem Biol 9 431-440 (2005).

Grozinger, C.M., Chao, E.D., Blackwell, H.E., et al. Identification of a class of small molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening. J Biol Chem 276(42) 38837-38843 (2001).

Westphal, C.H., Dipp, M.A., and Guarente, L. A therapeutic role for sirtuins in diseases of aging? Trends Biochem Sci 32(12) 555-560 (2007).

Yamamoto, H., Schoonjans, K., and Auwerx, J. Sirtuin functions in health and disease. Mol Endocrinol 21(8) 1745-1755 (2007).

Kaeberlein, M., McDonagh, T., Heltweg, B., et al. Substrate-specific activation of sirtuins by resveratrol. J Biol Chem 280(17) 17038-17045 (2005).

Tanner, K.G., Landry, J., Sternglanz, R., et al. Silent information regulator 2 family of NAD-dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose. Proc Natl Acad Sci USA 97(26) 14178-14182 (2000).

Cheung, W.L., Briggs, D.B., and Allis, C.D. Acetylation and chromosomal functions. Curr Opin Cell Biol 12 326-333 (2000 Jan 1).

Strahl, B.D., and Allis, D. The language of covalent histone modifications. Nature 403 41-45 (2000 Jan 6).

Show all 20 Hide all but first 3

10010991-96WELL

SIRT1 FRET Assay Buffer (10X)

1 ea

SIRT1 (human recombinant) FRET Assay Reagent

1 ea

SIRT1 FRET Peptide

2 × 1 ea

SIRT1 FRET NAD+

1 ea