CycLex SIRT1/Sir2 Deacetylase Fluorometric Assay Kit Ver.2

Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and nematode. In 2000, it was reported that the yeast Sir2 protein is a NAD(+)-dependent histone deacetylase that plays a critical role in transcriptional silencing, genome stability and longevity. A human homologue of Sir2, SIRT1, also functions as a NAD(+)-dependent-p53 deacetylase as well as a NAD(+)-dependent histone deacetylase. SIRT1 was shown to regulate the activity of the p53 tumor suppressor and inhibits apoptosis. These results have significant implications regarding an important role for SIRT1 in modulating the sensitivity of cells in p53-dependent apoptotic response and the possible effect in cancer therapy. Since the function of p53 is made to strengthen powerfully by using together with DNA damaging reagent, it is expected that inhibitor of SIRT1 becomes an effective anticancer drug. However, the conventional method for measuring SIRT1/Sir2 activity is very complicated and laborious. In order to measure SIRT1/Sir2 enzyme activity, it is necessary to prepare radioactive acetylated histone as a substrate. First, cells have to be labeled metabolically with radioactivity by adding radioactive acetic acid to the culture medium. Second, radioactive acetylated histone has to be purified from the cells. Following the reaction, it is necessary to extract and separate the radioactive acetyl group, which has been released from acetylated histone, using ethyl acetate to measure the activity of the enzyme based on the radioactivity. Although a method for measuring the activity of deacetylase without the use of radioactive substances was reported in recent years, owing to the use of fluorescent-labeled acetylated lysine as a substrate, the reaction product must be separated from the intact substrate and the fluorescent intensity measured by reverse phase HPLC. As mentioned above, these measurement systems are difficult to adapt for processing many samples under a variety of conditions, because of their complicated operation. Thus a simple system for biochemical analysis as well as for inhibitor screening without the use of radioactive substances is preferred.

The CycLex Research Product CycLex SIRT1/Sir2 Deacetylase Fluorometric Assay kit detects SIRT1/Sir2 activity in lysates. Primarily, the CycLex Research Product CycLex SIRT1/Sir2 Deacetylase Fluorometric Assay kit is designed for the rapid and sensitive evaluation of SIRT1/Sir2 inhibitors or activators using crude SIRT1/Sir2 fraction or purified SIRT1/Sir2. Additionally, any cultured primary cell, cell line, or tissue homogenate can be assayed for SIRT1/Sir2 activity with the CycLex Research Product CycLex SIRT1/Sir2 Deacetylase Fluorometric Assay kit if the appropriate antibody direct against SIRT1 or Sir2 is used for immunoprecipitation.

SIRT1 Assay Buffer, Fluoro-Substrate Peptide (0.2 mM), Fluoro-Deacetylated Peptide (0.2 mM), NAD (2 mM), Developer, Recombinant SIRT1, Stop Solution

1 year

SIRT1/Sir2