The Epigenase™ HDAC Activity/Inhibition Direct Assay Kit is a complete set of optimized buffers and reagents for measuring the activity or inhibition of total HDAC enzymes in nuclear extracts or purified HDAC isoforms (HDACs 1-11) from a broad range of species such as mammals, plants, fungi, and bacteria, and in a variety of forms including, but not limited to, cultured cells and fresh and frozen tissues. The Epigenase™ HDAC Activity/Inhibition Direct Assay Kit is a further refinement of its predecessor, the EpiQuik™ HDAC Activity/Inhibition Assay Kit, a popular assay method for HDAC activity and inhibition. This latest method retains the simplicity, rapidness, high throughput capability, and non-radioactivity featured in the previous version, while offering the following advantages:
Back ground InformationAcetylation of epsilon amino group of specific lysine residue contained in core histones is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes. The acetylation of histone by histone acetyltransferases (HAT) seems to be of particular significance, as it is associated with active regions of the genome. In contrast, histone deacetylation by histone deacetylases (HDAC) leads to transcription repression. So far, at least 4 classes of HDACs have been identified. Class I HDACs include 1, 2, 3 and 8. Class II HDACs are comprised of 4, 5, 6, 7, 9 and 10. Class III enzymes, known as the sirtuins, require NAD+ cofactors and include SIRTs 1 through 7. Class IV enzymes, which contains only HDAC11, have features of both Class I and II.
HDACs are tightly involved in cell cycle regulation, cell proliferation, and development of human diseases including cancer, cardiovascular and pulmonary diseases, and neurological diseases. For example, in various tumor cells, HDAC enzymes are found to be overexpressed. In chronic obstructive pulmonary disease (COPD) HDACs are observed to be decreased. In Huntington’s disease, HDAC4 is critically involved. Detection of activity and inhibition of HDACs would be important in elucidating mechanisms of epigenetic regulation of gene activation and silencing and would benefit cancer diagnostics and therapeutics.
Principle & ProcedureThe Epigenase™ HDAC Activity/Inhibition Assay Kit contains all reagents necessary for the measurement of HDAC activity/inhibition. In this assay, an acetylated histone HDAC substrate is stably coated onto the microplate wells. Active HDACs bind to the substrate and removes acetyl groups from the substrate. The HDAC-deacetylated products can be recognized with a specific antibody. The ratio or amount of deacetylated products, which is proportional to the enzyme activity, can then be colorimetrically measured by reading the absorbance in a microplate spectrophotometer at 450 nm. The activity of the HDAC enzyme is proportional to the OD intensity measured.