The HDAC2 assay kit24 (link) was purchased from BPS Bioscience (San Diego, CA). The assays were carried out in black background. Each well contained a volume of 50 μL including 30 μL buffer (BPS Bioscience, Catalog No. 50031), 5 μL BSA (1 mg/mL, Sigma-Aldrich), 5 μL inhibitor (1000, 631, 398, 251, 158, and 100 μM), 5 μL HDAC2 (1.8 ng/μL, BPS Bioscience, Catalog No. 50002), and 5 μL HDAC substrate (200 μM, BPS Bioscience, Catalog No. 50037). Prior to adding substrate, the plate was pre-incubated at 37 °C for 30 minutes. Upon the addition of substrate the plate was incubated at the same temperature for another 30 minutes. The HDAC assay developer (50 μL, BPS Bioscience, Catalog No. 50030) was then added to each well and the plate was incubated for another 15 minutes. The fluorescence was measured at excitation and emission wavelengths of 360 nm and 460 nm, respectively. We had the negative control containing protein without inhibitors and the positive control with TSA inhibitor. In addition, the readout of blank well containing no inhibitor and protein was subtracted from each well in measurement. All the assays were performed in triplicate.
Hdac2
Manufactured by BPS Biosciences
Sourced in United States
HDAC2 is a recombinant enzyme that catalyzes the deacetylation of histone proteins. It is a member of the histone deacetylase (HDAC) family of enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Hdac6, by BPS Biosciences (6 mentions)
Hdac3, by BPS Biosciences (6 mentions)
Hdac8, by BPS Biosciences (4 mentions)
Hdac1, by BPS Biosciences (3 mentions)
Hdac5, by BPS Biosciences (3 mentions)
Hdac4, by BPS Biosciences (3 mentions)
Recombinant human hdac1, by BPS Biosciences (2 mentions)
Synergy mx, by Agilent Technologies (2 mentions)
Hdac11, by BPS Biosciences (2 mentions)
Hdac9, by BPS Biosciences (2 mentions)
Hdac7, by BPS Biosciences (2 mentions)
Hdac assay developer, by BPS Biosciences (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Prism software, by GraphPad (1 mentions)
His tag monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Hdac glo 1 2 reagents, by Promega (1 mentions)
Synergy plate reader, by Agilent Technologies (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Celltiter glo reagent, by Promega (1 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
9 protocols using hdac2
1
HDAC2 Enzyme Inhibition Assay
2
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).
3
HDAC Enzyme Characterization Protocol
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Assays were performed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer [50 mM HEPES/Na, 100 mM KCl, 0.001% (v/v) tween-20, 0.2 mM tris(2-carboxyethyl)phosphine (TCEP), 0.5 mg/mL bovine serum albumin (BSA), pH 7.4] unless otherwise stated. Recombinant HDAC enzymes employed in µSPOT, inhibitor, and substrate assays were from commercial sources: HDAC1 (full length, C-terminal His tag, C-terminal FLAG tag, BPS Bioscience, cat. #: 50051, lots 170105-1 and 181108-1, purity ≥ 79%, activity ≥ 460 pmol/min/µg), HDAC2 (full length, C-terminal His tag, BPS Bioscience, cat. #: 50002, lots 160701 and 160630, purity ≥ 84%, activity ≥ 675 pmol/min/µg), HDAC3/NCoR2 (full length, C-terminal His tag, NCoR2 N-terminal GST tag, BPS Bioscience, cat. #: 50003, lots 130819 and 190327, purity ≥ 80%, activity ≥ 3000 pmol/min/µg), HDAC8 (full length, C-terminal His tag, BPS Bioscience, cat. #: 50008, lots 150714 and 161216, purity ≥ 90%, activity ≥ 300 pmol/min/µg), HDAC4 (aa 627‒1084, BPS Bioscience, cat. #: 50004, lot 130828-G, purity ≥ 89%, activity ≥ 103225 pmol/min/µg), HDAC6 (full length, BPS Bioscience, cat. #: 50056, lot 151130-C, purity ≥ 88%, activity ≥ 150 pmol/min/µg). Antibodies: 6x-His tag monoclonal antibody (HIS.H8), HRP-conjugated (ThermoFisher, cat. #: MA1-21315-HRP).
4
Enzymatic Inhibition of HDAC1-3 by UF010
5
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).
6
In Vitro HDAC Inhibition Assay
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The in vitro HDACs inhibitory assay of all compounds was performed by Sundia MediTech Company, Ltd. (Shanghai, China). It was conducted by fluorescent assay. Briefly, all compounds were serially diluted to certain concentrations. Then, the HDAC1 enzymes (Active Motif, 31504), HDAC2 (BPS bioscience, 50002), HDAC5 (BPS bioscience, 50005), HDAC6 (BPS bioscience, 50006), HDAC8 (Active Motif, 31566), HDAC11 (BPS bioscience, 50011), and compounds solution were dissolved in 1×Assay buffer, which was then added into a 384-well microplate. It was mixed briefly with gentle centrifuging and the plate was incubated at r.t. Substrate peptide and Trypsin mixture were added to each well, shaken for 1 minute, and the 384-plate was placed in a BioTek Synergy plate reader for data collection.
7
High-Throughput Screening of Epigenetic Modulators
8
Histone Deacetylase Assays and Immunoblotting
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Dulbecco's modified Eagle's medium (DMEM) with L-glutamine was purchased from GenDEPOT (Barker, TX), fetal bovine serum was purchased from HyClone, and penicillin and streptomycin were purchased from Gibco BRL (Gaithersburg, MD). Antibodies for α-tubulin, Ac-α-tubulin (Lys40), Histone H3, Ac-Histone H3 (Lys9), HDAC1, HDAC6, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Boston, MA). Goat anti-rabbit IgG horseradish peroxidase conjugate was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cell Titer 96 Aqueous One Solution cell proliferation assay kit was purchased from Promega (Madison, WI). Amersham ECL Select western blotting Detection Reagent was purchased from GE Healthcare. HDAC fluorogenic assay kits (HDAC1, #50061; HDAC2, #50062; HDAC3, #50073; HDAC4, #50064; HDAC6, #50076; HDAC11, #50687) were bought from BPS Bioscience (San Diego, CA).
9
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).
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