CycLex HDAC8 Deacetylase Fluorometric Assay Kit Ver.2

HDAC proteins are vital regulators of fundamental cellular events, including cell cycle progression, differentiation, and tumorigenesis (1, 2). A small-molecule inhibitor of HDAC, trichostatin A (TSA), arrests mammalian cells in both G1 and G2 (3, 4), while overexpression of HDAC1 in mouse cells reduces their growth rate by lengthening the duration of G2 and M (5). TSA induces terminal differentiation of mouse erythroleukemia cells and apoptosis of lymphoid and colorectal cancer cells. In addition, TSA treatment of cells expressing the PML zinc finger protein derepresses transcription and allows cells to differentiate normally (6). With this precedent, HDAC inhibitors are being actively explored as potential agents for the treatment of certain forms of cancer (7-9). The human HDACs are organized into three different classes based on their similarity to yeast HDAC proteins (1, 2). Class I enzymes are ubiquitously expressed and include HDAC1, -2, -3, and -8, which are homologous to the yeast RPD3 protein. Class II includes HDAC4, -5, -6, -7, -9, and -10, which are similar to yeast HDA1 and are expressed in a tissue-specific manner. The Sir2-like class III HDACs, including SIRT1 to -7, require NAD(+) for enzymatic activity. It has been reported that HDAC8 is important for the growth of human tumor cell lines and has a distinct inhibition pattern that differs from that of HDAC1 and -3, which both share 43% sequence identity with HDAC8. These findings lead to open the way to the development of selective inhibitors of this subtype as potential novel anticancer therapeutics. However, the conventional method for measuring HDAC activity is very complicated and laborious. In order to measure HDAC 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.

This kit is designed for the rapid and sensitive evaluation of HDAC inhibitors using recombinant HDAC8.

HDAC Assay Buffer, Fluoro-Substrate Peptide (0.2 mM), Fluoro-Deacetylated Peptide (0.2 mM), Trichostatin A (200 uM), Developer, Recombinant HDAC8, Stop Solution

1 year

HDAC8