Histone deacetylases (HDACs) are a family of nuclear enzymes which remove the acetyl group from the sidechains of lysine residues in histones. As such, they are important in chromatin remodeling and the level of acetylation is related to transcriptional activity. Acetylation, carried out by histone acetyltransferase, has been shown to induce an open conformation that allows transcription machinery access to promoters, while deacetylation by HDACs correlates with gene silencing. HDACs may represent a new target for anti-cancer therapy, since several studies have found that HDAC levels are altered in different types of cancer cells, and inhibitors have been shown to block cell growth and differentiation . HDACs are also believed to be responsible for the acetylation of non-histone proteins.
The mammalian HDACs, which have yeast homologs, have been grouped into three classes, but only classes I and II are thought to be targets for anti-cancer drugs. Using recombinant DNA technology, several of these enzymes have been expressed primarily in insect cells and less often in bacteria. Our interest was to produce large amounts of enzyme (in E.coli) for structural studies and to use in screening for inhibitors. We used this assay kit to monitor expression levels of several different protein constructs.
The assay utilizes a short peptide with an epsilon-acetylated lysine residue at C-terminal conjugated to the fluorophore 7-amino 4-methylcoumarin through the carboxyl group. The assay is carried out in two steps in a microtiter plate. First, samples of interest are incubated with the peptide to allow deacetylation (30-60 min). Second, the “Activator Solution” (trypsin) is added which will cleave the fluorophore from the deacetylated peptide, but not from the remaining acetylated substrate (15 min). A standard curve is generated from serial dilutions of the unacetylated peptide which are incubated with Activator Solution. The plate is then read in a fluorescent plate reader. The dye has an excitation max of 353 nm and an emission max of 448 nm, so you may need to purchase the appropriate filters, depending on your fluorometer. This will probably cost several hundred dollars.
Using this kit, I was unable to detect background HDAC activity in extracts of E.coli which did not express the recombinant protein. Using insect cells, some activity was seen in control cell extracts, but this was significantly lower than in cells expressing the recombinant protein. In conclusion, the kit was certainly adequate for expression studies. It is certainly suitable for studies requiring the measurement of HDAC activity levels in mammalian cells since only 4 ug of HeLa cell nuclear extract protein was recommended in control experiments described in the manual.
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Lloyd Waxman, PhD
Research Scientist
Brighter Ideas, Inc.
United States
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