Product Overview :
The HDAC Colorimetric Assay/Drug Discovery Kit is a complete assay system designed to measure histone deacetylase (HDAC) activity in cell or nuclear extracts, immunoprecipitates or purified enzymes. It comes in a convenient 96-well format, with all reagents necessary for Colorimetric HDAC activity measurements and calibration of the assay. In addition, a HeLa nuclear extract, rich in HDAC activity, is included with the kit. The extract is useful as either a positive control or as the source of HDAC activity for inhibitor/drug screening. Also included is the potent HDAC inhibitor, Trichostatin A, which may be used as model inhibitor.
The HDAC Colorimetric Assay/Drug Discovery Kit is based on the unique Substrate and Developer combination. The system (Colorimetric Histone deAcetylase Lysyl Substrate/Developer) is a highly sensitive and convenient alternative to radiolabeled, acetylated histones or peptide/HPLC methods for the assay of histone deacetylases. The assay procedure has two steps. First, the Substrate, which comprises an acetylated lysine side chain, is incubated with a sample containing HDAC activity (HeLa nuclear or other extract, purified enzyme, bead-bound immunocomplex, etc.). Deacetylation of the substrate sensitizes the substrate so that, in the second step, treatment with the Developer causes an increase in yellow intensity and absorption at 405nm. There is a linear correlation between the absorption and the amount of dye released within instrument limitations.
Description :
Histones form the protein core of nucleosomes, the DNA/protein complexes that are the subunits of eukaryotic chromatin. The histones’ N-terminal "tails" are subject to a variety of posttranslational modifications, including phosphorylation, methylation, ubiquitination, ADP-ribosylation and acetylation. These modifications have been proposed to constitute a 'histone code' with profound regulatory functions in gene transcription1. The best studied of these modifications, acetylation of the ε-aminos of specific histone lysine residues, are catalyzed by histone acetyltransferases (HATs). Histone deacetylases (HDACs) are responsible for hydrolytic removal of these acetyl groups.
Applications :
Colorimetric detection, HTS
Stability :
Store all components except the microtiter plate at -70°C for the highest stability. The HeLa Nuclear Extract must be handled with particular care in order to retain maximum enzymatic activity. Defrost it quickly in a RT water bath or by rubbing between fingers, then immediately store on an ice bath. The remaining unused extract should be refrozen quickly, by placing at -70°C. If possible, snap freeze in liquid nitrogen or a dry ice/ethanol bath. To minimize the number of freeze/thaw cycles, aliquot the extract into separate tubes and store at -70°C. The Substrate, when diluted in Assay Buffer, may precipitate after freezing and thawing. It is best, therefore, to dilute only the amount needed to perform the assays of that day.
Kit Components :
1. Nuclear Extract from HeLa Cells (human cervical cancer cell line)
STORAGE: -70°C; AVOID FREEZE/THAW CYCLES!
QUANTITY: 500µl
2. Substrate
FORM: 50mM in DMSO (dimethylsulfoxide)
STORAGE: -70°C
QUANTITY: 50µl
3. Developer Concentrate (20x)
FORM: 20x Stock Solution; Dilute in HDAC Assay Buffer before use.
STORAGE: -70°C
QUANTITY: 300µl
4. Trichostatin A (HDAC Inhibitor)
FORM: 0.2mM in DMSO (dimethylsulfoxide)
STORAGE: -70°C
QUANTITY: 100µl
5. Deacetylated Standard
FORM: 10mM in DMSO (dimethylsulfoxide)
STORAGE: -70°C
QUANTITY: 30µl
6. HDAC ASSAY BUFFER (50mM Tris/Cl, pH 8.0, 137mM NaCl, 2.7mM KCl, 1mM MgCl2)
STORAGE: -70°C
QUANTITY: 20ml
7. 1/2 VOLUME MICROPLATE
1 clear, 96-well
STORAGE: Room temperature
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