The cellular HDAC assay is based on the publications by Heltweg and Jung [36 (link)], Ciossek et al. [37 (link)] and Bonfils et al. [38 (link)] Minor modifications were still made as described in [39 (link)]. In 96-well plates (Corning, Kaiserslautern, Germany), 25,000 c/w THP-1 were seeded in 90 µL culture medium. After 24 h, increasing concentrations of YAK540 and TMP269 were added and incubated for an additional 18 h. Then, the reaction was started by adding 10 µL of 3 mM Boc-Lys(Ac)-AMC or 1 mM Boc-Lys(Tfa)-AMC (Bachem, Bubendorf, Switzerland) to achieve a final concentration of 0.3 mM Boc-Lys(Ac)-AMC or 0.1 mM Boc-Lys(Tfa)-AMC, respectively. After three hours of incubation under culture conditions, 100 µL/well stop buffer (25 mM Tris-HCl, pH 8.0, 127 mM KCl, 1 mM MgCl2, 1% NP40, 2 mg/mL trypsin, 10 µM panobinostat) was added. After another three hours under culture conditions, fluorescence intensity was measured at an excitation wavelength of 320 nm and an emission wavelength of 520 nm in the NOVOstar microplate reader (BMG LabTech, Offenburg, Germany).
Boc lys tfa amc
Manufactured by Bachem
Sourced in Switzerland
Boc-Lys(TFA)-AMC is a chemical compound commonly used in biochemical research. It is a protected lysine derivative with a trifluoroacetyl (TFA) group on the side chain and an aminomethylcoumarin (AMC) moiety. This compound can be utilized as a fluorogenic substrate for the detection and measurement of enzymatic activity.
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Lab products found in correlation
Boc lys ac amc, by Bachem (2 mentions)
Novostar microplate reader, by BMG Labtech (2 mentions)
96 well plate, by Corning (2 mentions)
Hdac8, by Bachem (1 mentions)
Prism 6, by GraphPad (1 mentions)
Pherastar fs, by BMG Labtech (1 mentions)
Black 96 well microtiter plate, by Greiner (1 mentions)
Pherastar optima, by BMG Labtech (1 mentions)
6 protocols using boc lys tfa amc
1
Cellular HDAC Assay Protocol
2
Measurement of HDAC Enzyme Activity
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All human recombinant enzymes were purchased from Reaction Biology Corp. (Malvern, PA, USA). The HDAC activity assay HDAC2 (cat nr. KDA-21-277), HDAC4 (cat nr. KDA-21-279), HDAC6 (cat nr. KDA-21-213), and HDAC8 (cat nr. KDA-21-481) was performed in 96-well-plates (Corning, Kaiserslautern, Germany). Briefly 20 ng of HDAC2/8, 17.5 ng of HDAC6 and 2 ng of HDAC4 per reaction were used. Recombinant enzymes were diluted in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 1 mg/mL BSA). After a 5 min incubation step the reaction was started with 10 µL of 300 µM (HDAC2), 150 µM (HDAC6) Boc-Lys(Ac)-AMC (Bachem, Bubendorf, Switzerland) or 100 µM (HDAC4), 60 µM (HDAC8) Boc-Lys-(TFa)-AMC (Bachem, Bubendorf, Switzerland). The reaction was stopped after 90 min by adding 100 µL stop solution (16 mg/mL trypsin, 2 µM panobinostat for HDAC2/6/8, 2µM CHDI0039 (kindly provided by the CHDI Foundation Inc., New York, USA) for HDAC4 in 50 mM Tris-HCl, pH 8.0, and 100 mM NaCl. 15 min after the addition of the stop solution the fluorescence intensity was measured at excitation of 355 nm and emission of 460 nm in a NOVOstar microplate reader (BMG LabTech, Offenburg, Germany).
3
Fluorometric Assay for HDAC Enzyme Activity
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The enzyme activity of HDACs was measured using the 4-methylcoumarin-7-amide (AMC), a fluorophore conjugated to acetylated lysine substrates, Boc-Lys(Ac)-AMC (Cat # K-6405, Aurum Pharmatech) or (Boc-Lys(Tfa)-AMC (Cat # I-1985, Bachem), as described previously.4 (link),25 Retina extract was prepared using lysis buffer (50 mM Tris-base, 10 mM EDTA, 0.5 mM sodium orthovanadate, 0.5% sodium deoxycholic acid, 1% Triton X-100, and cocktail protease inhibitor). Equal amounts of retina protein extract were incubated with 100 µM substrate in HDAC buffer (50 mM Tris-Cl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 0.1 mg/mL bovine serum albumin) in 96-well black bottom plates at room temperature for 1 hour. For inhibition of total HDAC activity, retina extracts were pre-incubated with Trichostatin A (1µM) before adding the substrates. Baseline fluorescence was determined before the addition of peptidase enzyme, trypsin. Following incubation, trypsin was added in excess (5 µg/reaction) to cleave the conjugated AMC to fluoresce. The amount of fluorogenic AMC generated was measured with a standard fluorospectrometer (Ex/Em at 355/460). The Boc-Lys(Ac)-AMC is a specific substrate to class I and IIb HDAC 1, 2, 3, and 6; whereas Boc-Lys(Tfa)-AMC is a specific substrate for class IIa and IV HDAC 4, 5, 7, 8, 9, 10. and 11.
4
HDAC8 Enzyme Activity Assay
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The enzyme activity assay
was performed in assay buffer [25 mM Tris-HCl (pH 8.0), 50 mM NaCl,
and 0.001% (v/v) Pluronic F-68] in black half-area 96-well microplates
(Greiner Bio-One). For the initial screening, 10 nM HDAC8 was preincubated
with the indicated compounds at 250 μM for 2 h at 30 °C.
For IC50 determination, 10 nM HDAC8 (100 nM for the mutational
study) and 1 nM HDAC4 were preincubated with a serial dilution of
the indicated compounds for 1 h. The reaction was initiated by the
addition of 20 μM Boc-Lys(TFA)-AMC (Bachem). After substrate
conversion at 30 °C for 15 min for HDAC8 and 1 h for HDAC8 mutants
and HDAC4, the reaction was stopped by adding 1.67 μM suberoylanilide
trifluoromethylketone (SATFMK). The deacetylated substrate was cleaved
with 0.42 mg/mL trypsin to release fluorescent 7-amino-4-methylcoumarin
(AMC), which was detected with a microplate reader (PHERAstar FS or
BMG LABTECH) with fluorescence excitation at 360 nm and emission at
460 nm. IC50 values were calculated by generating dose–response
curves in GraphPad Prism 6 and fitting those to a four-parameter logistic
model.
was performed in assay buffer [25 mM Tris-HCl (pH 8.0), 50 mM NaCl,
and 0.001% (v/v) Pluronic F-68] in black half-area 96-well microplates
(Greiner Bio-One). For the initial screening, 10 nM HDAC8 was preincubated
with the indicated compounds at 250 μM for 2 h at 30 °C.
For IC50 determination, 10 nM HDAC8 (100 nM for the mutational
study) and 1 nM HDAC4 were preincubated with a serial dilution of
the indicated compounds for 1 h. The reaction was initiated by the
addition of 20 μM Boc-Lys(TFA)-AMC (Bachem). After substrate
conversion at 30 °C for 15 min for HDAC8 and 1 h for HDAC8 mutants
and HDAC4, the reaction was stopped by adding 1.67 μM suberoylanilide
trifluoromethylketone (SATFMK). The deacetylated substrate was cleaved
with 0.42 mg/mL trypsin to release fluorescent 7-amino-4-methylcoumarin
(AMC), which was detected with a microplate reader (PHERAstar FS or
BMG LABTECH) with fluorescence excitation at 360 nm and emission at
460 nm. IC50 values were calculated by generating dose–response
curves in GraphPad Prism 6 and fitting those to a four-parameter logistic
model.
5
HDAC8 Enzyme Activity Assay
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Enzyme activity assay was executed in assay buffer (25 mM Tris-HCL pH 8.0, 50 mM NaCl, and 0.001 % (v/v) pluoronic F-68) in half area 96-well black microplates (Greiner Bio-One, Solingen, Germany). For IC50 determination, 10 nM HDAC8 was preincubated for 1 h with a serial dilution of the indicated compounds. The enzyme reaction was initiated by the addition of 20 µM Boc-Lys(TFA)-AMC (Bachem, Bubendorf, Switzerland). After substrate conversion at 30 °C for 1 h, the reaction was stopped by adding 1.67 µM suberoylanilide trifluoromethylketone (SATFMK). The deacetylated substrate was cleaved with 0.42 mg/mL trypsin to release fluorescent 7-amino-4-methylcoumarin (AMC, Amsterdam, The Netherlands), which was detected with a microplate reader (PHERAstar FS or BMG LABTECH) with fluorescence excitation at 360 nm and emission at 460 nm. IC50 values were calculated by generating dose-response curves in GraphPad Prism, and fitting those to a 4-parameter fit model.
6
Kinetic Characterization of KDAC8 Enzyme
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The measurements were carried out in a black 96-well microtiter plate (Greiner) at 30°C in assay buffer (see above). A serial two-fold dilution ranging from 50 µmol/L substrate, Boc-Lys(TFA)-AMC (Bachem), to 780 nmol/L, was prepared in an assay buffer containing 0.1 mg/mL trypsin. The enzymatic reaction was initiated by the addition of 10 nmol/L KDAC8. The release of fluorogenic AMC was observed in a micro plate reader (PHERAstar Optima, BMG Labtech) at 450 nm (Ex: 350 nm). The data points were plotted against time and blank corrected. For the determination of Michaelis-Menten parameters, a linear regression analysis of the initial slope (vi) of each curve was performed. Slopes were transformed into rates of AMC product formation using a calibration curve with different AMC concentrations. The obtained enzyme conversion rates were plotted against the respective substrate concentration and fitted to the Michaelis-Menten function using GraphPad Prism software. The turnover number (kcat) was calculated as
where vi is the substrate conversion rate and E is the enzyme concentration. All solutions were pre-incubated at 30°C.
where vi is the substrate conversion rate and E is the enzyme concentration. All solutions were pre-incubated at 30°C.
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