Apo-HDAC8-Bpa (10µM) was incubated with Zn2+ at a stoichiometric ratio on ice for 1 hour prior to dilution into the assay. The catalytic activity of HDAC8-Bpa mutants was measured using the Fluor-de-Lys tetrapeptide substrate Ac-Arg-His-Lys(ac)-Lys(ac)-aminomethylcoumarin (Enzo Life Sciences). Activity assays were run at 30°C in 1x assay buffer (25 mM HEPES pH 7.8, 137 mM NaCl and 3 mM KCl) that contained 100µM peptide and 1µM HDAC8-Bpa mutant. The ratio of product fluorescence (ex, = 340 nm, em. = 450 nm) divided by the substrate fluorescence (ex. = 340 nm, em. = 380 nm) increases with product concentration. The concentration of product was determined using a standard curve and the initial rate was determined from the product formed over time. The catalytic (kcat/KM,app) was determined from dividing the initial rate by the substrate and enzyme concentrations, assuming a linear fit.
Fluor de lys
Manufactured by Enzo Life Sciences
Sourced in United States
The Fluor-de-Lys is a fluorometric assay product developed by Enzo Life Sciences. It is designed to quantitatively measure the activity of specific enzymes. The product provides a simple, sensitive, and reliable method for detecting and analyzing enzyme activity in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Developer solution, by Enzo Life Sciences (1 mentions)
Fluor de lys kit, by Enzo Life Sciences (1 mentions)
Gemini xs, by Molecular Devices (1 mentions)
Flx800 microplate reader, by Agilent Technologies (1 mentions)
Prism software, by GraphPad (1 mentions)
Clariostar fluorimeter, by BMG Labtech (1 mentions)
Biomol, by Enzo Life Sciences (1 mentions)
9 protocols using fluor de lys
1
Kinetic analysis of HDAC8-Bpa mutants
2
HDAC Inhibitor Assay in HeLa Cells
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Human cervical cancer, HeLA,
cells were grown in DMEM supplemented with 1× Pen-strep solution
and 10% FBS and incubated at 37 °C. The cells were subcultured
twice a week. The cells were harvested when 80% confluent and lysed
in IP lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol,
0.5% Triton X-100, 1× Protease Inhibitor Cocktail) to isolate
total proteins. Antibody (2 μg) (HDAC8, HDAC4, and HDAC6) is
incubated with 60 μL of Dyna beads for 30 min on rotation. Then,
100 μg of total protein was added to the bead–antibody
complex and incubated for 4 h at 4 °C on rotation. The beads
were washed with lysis buffer and used as enzyme source for the activity
assay. For HDAC activity assay,46 (link) the bead-bound
enzyme was incubated with HDAC substrate, Fluor-de-lys (Enzo Life
Sciences), and different concentrations (10, 1, 0.1, 0.01, and 0.001
μM) of compound5 for 15 min, followed by addition
of developer solution according to the manufacturer’s protocol
(Enzo Life Sciences), and the fluorescence was measured at 360 and
460 nm excitation and emission wavelengths, respectively. The % HDAC
enzyme activity is calculated as a ratio to vehicle-treated control.
cells were grown in DMEM supplemented with 1× Pen-strep solution
and 10% FBS and incubated at 37 °C. The cells were subcultured
twice a week. The cells were harvested when 80% confluent and lysed
in IP lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol,
0.5% Triton X-100, 1× Protease Inhibitor Cocktail) to isolate
total proteins. Antibody (2 μg) (HDAC8, HDAC4, and HDAC6) is
incubated with 60 μL of Dyna beads for 30 min on rotation. Then,
100 μg of total protein was added to the bead–antibody
complex and incubated for 4 h at 4 °C on rotation. The beads
were washed with lysis buffer and used as enzyme source for the activity
assay. For HDAC activity assay,46 (link) the bead-bound
enzyme was incubated with HDAC substrate, Fluor-de-lys (Enzo Life
Sciences), and different concentrations (10, 1, 0.1, 0.01, and 0.001
μM) of compound
of developer solution according to the manufacturer’s protocol
(Enzo Life Sciences), and the fluorescence was measured at 360 and
460 nm excitation and emission wavelengths, respectively. The % HDAC
enzyme activity is calculated as a ratio to vehicle-treated control.
3
Quantifying HDAC Activity in bMECs
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Plates of 96 wells were used to grow 1 × 105 bMECs with the combined hormones for 6 or 12 h, and then were infected with S. aureus for 2 h. HDAC activity was measured using a Fluor de Lys® kit (Enzo LifeSciences, Farmingdale, NY, USA) to analyze class I HDACs (HDAC1, 2, 3 and 8), IIa (HDAC4, 5, 7 and 9) and IIb (HDAC6 and 10). Then, bMECs were incubated with 2000 pmol of the acetylated Fluor de Lys® substrate for 4 h. Cells, media and the standard curve of the deacetylated substrate were prepared according to the manufacturer’s directions; relative fluorescence unit (RFU) counts were acquired with a Varioskan (Enzo LifeSciences) plate reader (360 nm excitation, 460 nm emission). A standard curve was generated using serial 1:10 dilutions of the deacetylated Fluor de Lys standard and developer supplied with the BML-AK503 HDAC fluorometric cellular activity assay kit. Trichostatin (TSA, 1 μM) was purchased from Sigma, and was used as an inhibitor of HDACs. HDAC activity was calculated, considering the effect of vehicle-treated bMECs as basal activity (normalized to onefold). According to the manufacturer’s instructions, the assays were run in duplicate from two different experiments.
4
Fluorometric HDAC Activity Assay
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HDAC activity assay was determined using the fluorometric in vitro HDAC activity assay FLUOR DE LYS® (Enzo Life Sciences, Inc.) according to manufacturer’s protocol, and background subtracted relative fluorescence unit (RFU) counts were acquired by fluorometer Molecular Devices, GeminiXS (360 nm excitation, 450 nm emission). In this experiment the inhibitors were used as follows: 5 nM IN01, 5 nM IN04, 5 nM IN14, 0.5 nM TSA and 5 nM PB. To minimize variability the inhibition assays were performed as technical triplicate.
5
In vitro HDAC Deacetylase Activity Assay
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In vitro deacetylase activity was detected using the fluorophore release from adequate Fluor de Lys acetylated substrates upon deacetylase enzymatic activity (reagents purchased from Enzo Life Sciences, enzymes purchased from Sigma Aldrich or produced by the Reaction Biology Corporation, Malvern, PA, using the Reaction Biology HDAC Spectrum platform, www.reactionbiology.com ). HeLa nuclear extracts were used as source of class I HDACs. Trichostatin A (a pan-HDAC inhibitor) was used as positive control. Compounds were tested in dose-response (2% DMSO, in HDAC assay buffer). For each tested concentration, a corresponding blank was included to detect fluorescence artefacts. Assays were conducted in duplicate in 96-well plates at 37 °C for 30 (HeLa nuclear extracts) or 60 (HDAC2, 8, 6 and 4) min. Enzymatic reactions were stopped by adding HDAC developer I or II (Enzo Life Sciences) to each well, and the plate was incubated at room temperature. The resulting fluorescence intensity was measured at 360/460 nm or 485/530 nm, using a FLx800 microplate reader (Biotek). Untreated enzyme wells were included in each experiment as the 100% deacetylase activity control. IC50 values were determined through a dose-response assay and plotted on GraphPad Prism 6.0.
6
Fluorometric Assay for KDAC Activity
7
Deacetylation Assay for HDAC6 Variants
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Deacetylation activity of HDAC6 variants was determined using acetyl-Gly-Ala-(acetyl-Lys)-AMC (GAK(Ac) -AMC; Bachem, Bubendorf, Switzerland), Boc-(acetyl-Lys)-AMC (Boc-K(Ac)-AMC; Bachem) and Fluor-de-Lys (Enzo Life Sciences, Plymouth Meeting, PA, USA) substrates. In general, a given HDAC6 variant was incubated with a substrate in a reaction buffer comprised of 50 mM HEPES, 140 mM NaCl, 10 mM KCl, 1 mg/ml bovine serum albumin (BSA), and 1 mM TCEP, at a pH 7.4 adjusted with NaOH (total volume 20 µl). This incubation occurred in 384-well plates for 30 min with vigorous shaking at 37 °C. The reaction was stopped by addition of 20 µl of trypsin solution (2 mg/ml trypsin, 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA; pH 7.4 adjusted with NaOH). Following the 15-min incubation at 37 °C, a fluorescence signal of released aminomethylcoumarin was quantified using a CLARIOstar fluorimeter (BMG Labtech GmbH, Ortenberg, Germany) with excitation/emission wavelengths set at 365/440 nm, respectively. The data were fitted using the GraphPad Prism software (GraphPad Software, San Diego, CA, USA) and kinetic values were calculated by non-linear regression analysis.
8
Fluorometric Assay of Sirtuin 1 Activity
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Sirtuin 1 activity in kidney homogenates was measured using a fluorometric assay kit (Fluor-de-Lys, Enzo Life Sciences, Farmingdale, NY, USA) following the manufacturer’s recommended instructions.
9
In Vitro Sirtuin Inhibition Analysis
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To a solution of 2-bromo-4,5-dimethoxyphenylacetic acid (0.25 g, 0.90 mmol) in THF (23 mL), HATU (0.39 g, 0.99 mmol) and DIPEA (0.31 mL, 1.8 mmol) were added and the new mixture was stirred for 30 min at rt. Then, the solution was cooled at 0 ºC and a solution of compound 14 (0.24 g, 0.90 mmol) in THF ( 12 To a solution of compound 16 (0.12 g, 0.24 mmol) in DMA (5. Analysis of in vitro sirtuin inhibition. SIRT1 and SIRT2 in Vitro Assay. The compounds were studied using the Fluor de Lys fluorescence assays which are described in the BioMol product sheet (Enzo Life Sciences). In assays the BioMol KI177 substrate was used for SIRT1 and the KI179 substrate for SIRT2. The determined Km value of SIRT1 for KI177 was 58 µM and the Km of SIRT2 for KI179 was 198 µM. 32 The Km values of SIRT1 and SIRT2 were 558 µM and 547µM for NAD+ reported by BioMol, respectively.
Briefly, assays were carried out using the Fluor de Lys acetylated peptide substrate at 0.7 Km and NAD+ (Sigma N6522 or BioMol KI282) at 0.9 Km, recombinant GST-SIRT1/2-enzyme and SIRT assay buffer (KI286). GST-SIRT1 and GST-SIRT2 were produced as described previously. 33, 34 The buffer together with Fluor de Lys acetylated peptide substrate, NAD+ and DMSO/compounds in DMSO (2.5 µL in 50 µL total reaction volume; DMSO from Sigma, D2650) were preincubated for 5 min at room temperature. Then enzyme was added to start the reaction.
Briefly, assays were carried out using the Fluor de Lys acetylated peptide substrate at 0.7 Km and NAD+ (Sigma N6522 or BioMol KI282) at 0.9 Km, recombinant GST-SIRT1/2-enzyme and SIRT assay buffer (KI286). GST-SIRT1 and GST-SIRT2 were produced as described previously. 33, 34 The buffer together with Fluor de Lys acetylated peptide substrate, NAD+ and DMSO/compounds in DMSO (2.5 µL in 50 µL total reaction volume; DMSO from Sigma, D2650) were preincubated for 5 min at room temperature. Then enzyme was added to start the reaction.
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