The in vitro inhibitory activity of 1a , 1g , 1l , 1q , 1u , 1v and SAHA against two human HDAC isoforms (1 and 6) were measured using a previously published protocol [55 (link)]. OptiPlate-96 black microplates (Perkin Elmer) were used with an assay volume of 50 µ L. 5 µ L test compound or control, diluted in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.1 mg/mL BSA), were incubated with 35 µ L of the fluorogenic substrate ZMAL (Z- (Ac)Lys-AMC) [56 (link)] (21.43 µM in assay buffer) and 10 µ L of human recombinant HDAC1 (BPS Bioscience, Catalog# 50051) or HDAC6 (BPS Bioscience, Catalog# 50006) at 37 °C. After an incubation time of 90 min, 50 µ L of 0.4 mg/mL trypsin in trypsin buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl) were added, followed by further incubation at 37 °C for 30 min. Fluorescence was measured with an excitation wavelength of 355 nm and an emission wavelength of 460 nm using a Fluoroskan Ascent microplate reader (Thermo Scientific). All compounds were evaluated in duplicate in at least two independent experiments.
Optiplate 96 black microplates
Manufactured by PerkinElmer
Sourced in United States
The OptiPlate-96 black microplates are a type of laboratory equipment used for various applications, such as cell-based assays, fluorescence-based assays, and luminescence-based assays. These microplates are designed with a black color, which helps to minimize background signal and improve the signal-to-noise ratio. The plates have a 96-well format, which is a common standard for high-throughput screening and other assay applications.
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8 protocols using optiplate 96 black microplates
1
HDAC Inhibition Assay: Screening Compounds
2
HDAC Inhibitory Activity Assay
3
HDAC Inhibition Activity Assay
4
HDAC1 and HDAC6 Inhibition Assay
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The inhibition of human HDAC1 and 6 was determined as previously described [50 (link)]. OptiPlate-96 black microplates (Perkin Elmer) were used with an assay volume of 50 µL. 5 µL test compound or control, diluted in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.1 mg/mL BSA), were incubated with 35 µL of the fluorogenic substrate ZMAL (Z-Lys(Ac)-AMC) (21.43 µM in assay buffer) and 10 µL of human recombinant HDAC1 (BPS Bioscience, Catalog# 50051) or HDAC6 (BPS Bioscience, Catalog# 50006) at 37 °C. After an incubation time of 90 min, 50 µL of 0.4 mg/mL trypsin in trypsin buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl) were added, followed by further incubation at 37 °C for 30 min. Fluorescence was measured with an excitation wavelength of 355 nm and an emission wavelength of 460 nm using a Fluoroskan Ascent microplate reader (Thermo Scientific). All compounds were tested at least twice and in duplicates, and the 50% inhibitory concentration (IC50) was determined by plotting dose response curves and nonlinear regression with GraphPad Prism.
5
HDAC1 and HDAC6 Inhibition Assay
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The inhibition of human HDAC1 and 6 was determined as previously described [35 (link)]. OptiPlate-96 black microplates (Perkin Elmer Inc., Waltham, MA, USA) were used with an assay volume of 50 µL. A total of 5 µL test compound or control, diluted in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.1 mg/mL BSA), was incubated with 35 µL of the fluorogenic substrate ZMAL (Z-Lys(Ac)-AMC) (21.43 µM in assay buffer) and 10 µL of human recombinant HDAC1 (BPS Bioscience Inc., San Diego, CA, USA, Catalog# 50051) or HDAC6 (BPS Bioscience Inc., San Diego, CA, USA, Catalog# 50006) at 37 °C. After an incubation time of 90 min, 50 µL of 0.4 mg/mL trypsin in trypsin buffer (50 mM Tris-HCl, pH 8.0, and 100 mM NaCl) was added, followed by further incubation at 37 °C for 30 min. Fluorescence was measured with an excitation wavelength of 355 nm and an emission wavelength of 460 nm using a Fluoroskan Ascent microplate reader (Thermo Scientific, Waltham, MA, USA). All compounds were tested at least twice and in duplicates, and the 50% inhibitory concentration (IC50) was determined by plotting dose response curves and nonlinear regression with GraphPad Prism (San Diego, CA, USA).
6
HDAC1 and HDAC6 Inhibition Assay
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The assays were performed according to a previously published procedure.82 (link) OptiPlate-96 black microplates (PerkinElmer) were used. The total assay volume was 60 μL. 52 μL of human recombinant HDAC1 (BPS Bioscience, catalogue no. 50051) or human recombinant HDAC6 (BPS Bioscience, catalogue no. 50006) in incubation buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 1 mg mL−1 BSA) were incubated with 3 μL of different concentrations of inhibitors in DMSO and 5 μL of the fluorogenic substrate ZMAL (Z-(Ac)Lys-AMC) (126 μM) for 90 min at 37 °C. After the incubation time, 60 μL of the stop solution, comprising 33 μM trichostatin A (TSA) and 6 mg mL−1 trypsin in trypsin buffer (Tris-HCl 50 mM, pH 8.0, NaCl 100 mM), was added. The plate was incubated again at 37 °C for 30 min, and fluorescence was measured on a BMG LABTECH POLARstar OPTIMA plate reader (BMG Labtechnologies, Germany) with an excitation wavelength of 390 nm and an emission wavelength of 460 nm.
7
HDAC1 and HDAC6 Inhibition Assay
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In vitro inhibitory activities against HDAC1 and HDAC6 were measured using a previously published protocol. 55 OptiPlate-96 black microplates (Perkin Elmer) were used with an assay volume of 50 µL.
8
Assessing HDAC1 and HDAC6 Inhibition
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In vitro inhibitory activities against HDAC1 and HDAC6 were measured using a previously published protocol. 41 OptiPlate-96 black microplates (Perkin Elmer) were used with an assay volume of 50 µL. 5 µL test compound or control, diluted in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.1 mg/mL BSA), were incubated with 35 µL of the fluorogenic substrate ZMAL (Z-Lys(Ac)-AMC) (21.43 µM in assay buffer) and 10 µL of human recombinant HDAC1 (BPS Bioscience, Catalog# 50051) or HDAC6 (BPS Bioscience, Catalog# 50006) at 37 °C. After an incubation time of 90 min, 50 µL of 0.4 mg/mL trypsin in trypsin buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl) were added, followed by further incubation at 37 °C for 30 min. Fluorescence was measured with an excitation wavelength of 355 nm and an emission wavelength of 460 nm using a Fluoroskan Ascent microplate reader (Thermo Scientific). All compounds were tested at least twice and in duplicates and the 50% inhibitory concentration (IC50) was determined by plotting dose response curves and nonlinear regression with GraphPad Prism.
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