HDAC fluorometric assay

Sowjanya Thatikonda; Venkatesh Pooladanda; Dilep Kumar Sigalapalli; Chandraiah Godugu

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HDAC fluorometric assay

ST Sowjanya Thatikonda

VP Venkatesh Pooladanda

DS Dilep Kumar Sigalapalli

CG Chandraiah Godugu

This method is extracted from research article: Cell Death Dis, Jan 2020

Piperlongumine regulates epigenetic modulation and alleviates psoriasis-like skin inflammation via inhibition of hyperproliferation and inflammation

DOI: 10.1038/s41419-019-2212-y

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HDAC inhibitory activity of PPL was measured by the Histone Deacetylase Assay Kit, Fluorometric (Sigma-Aldrich, USA), which was based on a two-step enzymatic reaction. Here various concentrations of PPL (0.05, 0.1, 1, and 2.5 µM) were incubated with the HeLa cell lysate and substrate having acetylated lysine residues and bound fluorescent groups, which were provided along with kit. After 30 min incubation, developer solution was added to the above components and further incubated for 10 min. Later, the fluorescence was measured with the multimode plate reader (Spectramax M4, Molecular Devices, USA). The HDAC inhibitor TSA was used as a standard to compare the HDAC inhibitory activity of PPL from the standard curve plotted from the non-acetylated substrate (standard), which was provided in the kit.

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