Hdac8

Manufactured by BPS Biosciences

Sourced in United States

HDAC8 is a recombinant enzyme product offered by BPS Biosciences. It is a histone deacetylase enzyme that catalyzes the removal of acetyl groups from lysine residues on histone proteins.

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5 protocols using hdac8

HDAC Inhibition Assay Protocol

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The assays were carried out by Shanghai ChemPartner Co., Ltd (Shanghai, China), according to our previous method [23 (link),24 (link)]. Briefly, different concentrations of compounds were incubated with recombinant human HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8 (BPS Biosciences, San Diego, CA, USA) at room temperature for 15 min, which was followed by adding Ac-peptide-AMC substrates to initiate the reaction in Tris-based assay buffer. Reaction mixtures were incubated at room temperature for 60 min in HDAC1, HDAC2, HDAC3 and HDAC6 assays, and were incubated for 240 min in HDAC8 assay. Then, the stop solution containing trypsin was added. The coupled reaction was incubated for another 90 min at 37 °C. Fluorescent AMC released from substrate was measured in SynergyMx (BioTek, Winooski, VT, US) using filter sets as excitation = 355 nm and emission = 460 nm. IC₅₀ values were calculated by GraphPad Prism software (7.0 version., GraphPad Software, San Diego, CA, USA).

Zhang B., Ruan Z.W., Luo D., Zhu Y., Ding T., Sui Q, & Lei X. (2020). Unexpected Enhancement of HDACs Inhibition by MeS Substitution at C-2 Position of Fluoro Largazole. Marine Drugs, 18(7), 344.

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HDAC Inhibition Assay Protocol

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The assays were carried out by Shanghai ChemPartner Co., Ltd. (Shanghai, China). Briefly, different concentrations of compounds were incubated with recombinant human HDAC1, HDAC2, HDAC3, HDAC6, and HDAC8 (BPS Biosciences, San Diego, CA, USA) at room temperature for 15 min, which was followed by adding Ac-peptide-AMC substrates to initiate the reaction in Tris-based assay buffer. Reaction mixtures were incubated at room temperature for 60 min in the HDAC1, HDAC2, HDAC3, and HDAC6 assays, and were incubated for 240 min in the HDAC8 assay. Then a stop solution containing trypsin was added. The coupled reaction was incubated for another 90 min at 37 °C. Fluorescent AMC released from substrate was measured in SynergyMx (BioTek, Winooski, VT, USA) using filter sets as excitation = 355 nm and emission = 460 nm. IC₅₀ values were calculated by GraphPad Prism version 4.00 Windows (GraphPad Software, San Diego, CA, USA).

Zhang B., Shan G., Zheng Y., Yu X., Ruan Z.W., Li Y, & Lei X. (2019). Synthesis and Preliminary Biological Evaluation of Two Fluoroolefin Analogs of Largazole Inspired by the Structural Similarity of the Side Chain Unit in Psammaplin A. Marine Drugs, 17(6), 333.

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High-Throughput Screening of Epigenetic Modulators

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Four cell lines (human colon cancer cell line HCT116, human embryonic kidney cell line HEK293, human liver cancer cell line HepG2, and human B lymphoma cell line SU-DHL-6), fetal bovine serum (FBS), Eagle's Minimum Essential Medium (EMEM), and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from American Type Cell Collection (ATCC, Manassas, VA, USA). H9-derived human neural stem cells, defined FBS, trypsin-EDTA, fibronectin, basal fibroblast growth factor (bFGF), epidermal growth factor (EGF), KnockOut™ Dulbecco's Modified Eagle Medium (DMEM)/F12, and penicillin-streptomycin, StemPro^® Neural Supplement were acquired from Life Technologies, Carlsbad, CA, USA. Chemicals and epigenetic compound libraries were purchased from Cayman Chemicals (Ann Arbor, MI), Santa Cruz Biotechnology (Dallas, TX, USA), SelleckChem (Houston, TX, USA), and Sigma-Aldrich (St. Louise, MO, USA). HDAC-Glo I/II and CellTiter-Glo reagents were purchased from Promega, Madison, WI. Fluorogenic assay kits for HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, and HDAC10 were acquired from BPS Bioscience (San Diego, CA). White solid bottom 1536-well assay plates were purchased from Greiner Bio-One (Monroe, NC).

Hsu C.W., Shou D., Huang R., Khuc T., Dai S., Zheng W., Klumpp-Thomas C, & Xia M. (2016). Identification of HDAC inhibitors using a cell-based HDAC I/II assay. Journal of biomolecular screening, 21(6), 643-652.

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HDAC Inhibitor Screening and Analysis

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Dulbecco’s Modified Eagle’s medium (DMEM) with L-glutamine was purchased from GenDEPOT (Barker, TX, USA) and RPMI 1640 medium, fatal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco BRL (Gaithersburg, MD, USA). Antibodies specific for α-tubulin, Ac-α-tubulin, Histone H3, Ac-Histone H3, PARP, caspase 3, cleaved caspase 8, β-actin HDAC1, and HDAC6 were purchased from Cell Signalling Technology (Boston, MA, USA). Rad52 antibody and goat anti-rabbit IgG horseradish peroxidase conjugate were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Cell Titre 96 Aqueous One Solution cell proliferation assay kit was purchased from Promega (Madison, WI, USA). Amersham ECL select Western blotting detection reagent was purchased from GE Healthcare (Waukesha, WI, USA). HDAC fluorogenic assay kits (HDAC1, HDAC3, HDAC6, and HDAC8) were purchased from BPS Bioscience (San Diego, CA, USA). OxiSelect™ Comet Assay Kit was purchased from Cell Biolabs, Inc (San Diego, CA, USA).

Song Y., Park S.Y., Wu Z., Liu K.H, & Seo Y.H. (2020). Hybrid inhibitors of DNA and HDACs remarkably enhance cytotoxicity in leukaemia cells. Journal of Enzyme Inhibition and Medicinal Chemistry, 35(1), 1069-1079.

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HDAC Enzyme Activity Assay Protocol

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Enzyme activity measurements of class I and IIa HDACs were performed using a cell‐free system and according to a method previously described.16 We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA). To test the enzyme activity of LMK235, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μmol/L concentrations were used. For the IC₅₀ calculations, every data point was normalized to the vehicle (100% activity). The normalized data were fitted with a Hill non‐linear curve fit (OrigionPro 9.0). Employing the “Find X from Y” function in OrigionPro 9.0 resulted in the IC₅₀ values (50% activity).

Choi S.Y., Kee H.J., Sun S., Seok Y.M., Ryu Y., Kim G.R., Kee S., Pflieger M., Kurz T., Kassack M.U, & Jeong M.H. (2019). Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension. Journal of Cellular and Molecular Medicine, 23(4), 2801-2812.

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