The DUB activity of purified PRRSV PLP2 was assayed in 50 μL reaction containing indicated amount of purified PLP2 protein, 1 μM Ub-AMC (Boston biochem, U-550), and 1× reaction buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, and 2 mM DTT) in a 96-well black microplate. The fluorescent intensity (excitation, 345nm, emission, 445nm) of each well was observed by infinite M1000 Pro plate reader (Tecan, Inc).
Ub amc
Manufactured by R&D Systems
Sourced in United States
Ub-AMC is a synthetic fluorogenic substrate for detecting and measuring the activity of deubiquitinating enzymes (DUBs). It consists of the ubiquitin protein conjugated to the fluorogenic molecule 7-amino-4-methylcoumarin (AMC). The cleavage of the Ub-AMC substrate by DUBs releases the AMC fluorophore, which can be detected using a fluorometer.
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Lab products found in correlation
Isg15 amc, by R&D Systems (4 mentions)
Ub amc, by Molecular Devices (3 mentions)
Ub vme, by R&D Systems (2 mentions)
Spectramax m2, by Molecular Devices (2 mentions)
Infinite m1000 pro fluorometer, by Tecan (2 mentions)
Ub amc, by PerkinElmer (2 mentions)
Envision plate reader, by PerkinElmer (2 mentions)
Bortezomib, by Cell Signaling Technology (2 mentions)
Anti ubiquitin p4d1, by Santa Cruz Biotechnology (2 mentions)
Ha ubiquitin vinyl sulfone ha ub vs, by R&D Systems (2 mentions)
Anti usp14 d8q6s, by Cell Signaling Technology (2 mentions)
Anti gapdh and anti ha tag, by Bioworld Technology (2 mentions)
Anti uchl5, by Abcam (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
7 amido 4 methylcoumarin, by Merck Group (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Infinite m1000 pro plate reader, by Tecan (1 mentions)
Proxiplate 384 plus f 384 shallow well microplate, by PerkinElmer (1 mentions)
Envision 2104 multilabel reader, by PerkinElmer (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
33 protocols using ub amc
1
Assay of PRRSV PLP2 Deubiquitinating Activity
2
Fluorogenic Assay for DUB Activity
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DUB activity was measured using the fluorogenic substrate 7-amino-4-methylcoumarin ubiquitin (Ub-AMC, Boston Biochem). 1 μM Ub-AMC was added to 100 nM protein samples in buffer 20 mM Tris pH 8.0, 0.1% BSA, 1 mM DTT. The concentration of isopeptidase T was 10 nM. Experiments were performed at 25°C. Ub-AMC cleavage was monitored as a function of time measuring the increased fluorescence emission at 460 nm after excitation at 380 nm. Experiments in the presence of free ubiquitin were performed in similar conditions, without adding BSA to the buffer. Cleavage of di-ubiquitin by JosK117-only and its mono-ubiquitinated form was performed at 25°C in 50 mM Tris pH 7.5, 0.5 mM DTT, using 20 μM Josephin and 20 μM K48- and K63-linked di-ubiquitin.
3
Fluorogenic Substrates for SARS-CoV-2 PLpro and Mpro
4
Enzymatic Activity Assay for UCHL1 and ZUFSP
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Ten microliters of 20 nM UCHL1 (final concentration 10 nM) or 500 nM ZUFSP-His6 (final concentration 250 nM) was added to 10 µL of 500 nM Ub-AMC (Boston Biochem; final concentration 250 nM) in PBS buffer (pH 7.4) containing 5 mM TCEP and 0.1 mg/mL bovine serum albumin (BSA) in a black ProxiPlate-384 Plus F 384-shallow well microplate (PerkinElmer). Fluorescence was detected on a Wallac EnVision 2104 Multilabel reader (PerkinElmer; excitation 355 nm, emission 460 nm) with reads interspaced by 10 s of shaking. The fluorescence reading from a negative control (Ub-AMC only) was subtracted from each value.
5
Fluorometric Assay of Uch-L1 Deubiquitinylation
6
In vitro Deubiquitinase Enzymatic Assay
7
Synthesis of SARS-CoV-2 Protease Substrates
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The SARS-CoV-2 PLpro FRET substrate Dabcyl-FTLRGG/APTKV(Edans) and the SARS-CoV-2 Mpro FRET substrate Dabcyl-KTSAVLQ/SGFRKME(Edans) were synthesized by solid-phase synthesis through iterative cycles of coupling and deprotection using the previously optimized procedure.27 (link) Ub-AMC and ISG15-AMC were purchased from Boston Biochem (catalog # U-550-050 and UL-553-050 respectively).
8
Fluorometric Assay for Atx3 Deubiquitinase Activity
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6His-atx3 produced in BL21(DE3)-SI cells was purified as described previously by our group (Gales et al., 2005 (link)). Incubation of protein samples (200 µM) with 0.5 µM Ub-AMC (Boston Biochem) was performed in the presence of 20 mM Hepes, pH 7.5, 5% glycerol, and 1 mM EDTA, 0.1 mg/ml BSA, and 10 mM DTT, in 96 well plates. Product formation was assessed at 30°C by fluorescence recording (excitation: 380 nm; emission: 460 nm). For each technical replicate, initial reaction velocity was calculated as the slope of the trend line traced based on the fluorescence values (relative fluorescence units) of the first 2 min of reaction, after subtraction of the value of the negative control lacking 6His-atx3 (with an additional 0.2 M BSA). The obtained velocity values were normalized to the mean WT velocity of the respective experiment.
Incubation of the samples (100 nM) with 250 nM K48- or K63-linked hexameric polyUb chains (Boston Biochem) was performed after mixing with 50 mM Hepes, 0.5 mM EDTA, 0.1 µg/ml ovalbumin, and 1 mM DTT and left to occur for 20 h, at 37°C. Samples (10 µl) were taken from the reaction mixture at 0, 2, 5, and 20 h and immediately denatured by adding 2× concentrated sample buffer.
Incubation of the samples (100 nM) with 250 nM K48- or K63-linked hexameric polyUb chains (Boston Biochem) was performed after mixing with 50 mM Hepes, 0.5 mM EDTA, 0.1 µg/ml ovalbumin, and 1 mM DTT and left to occur for 20 h, at 37°C. Samples (10 µl) were taken from the reaction mixture at 0, 2, 5, and 20 h and immediately denatured by adding 2× concentrated sample buffer.
9
Enzymatic Assay for USP21 Inhibition
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USP21 enzymatic assay was performed with the C-terminal derivatization of ubiquitin with 7-amido-4-methylcoumarin (Ub-AMC) (Boston Biochem). Inhibitory effects of the ubiquitin variants were measured in the assay buffer [50 mM Hepes (pH 7.5), 0.01% Tween 20, and 10 mM DTT] with 1 μM Ub-AMC, 15 nM USP21, and varying concentrations of the ubiquitin variants. USP21 and ubiquitin variants were mixed in the assay buffer and incubated for 30 min at room temperature. The reaction was started by the addition of Ub-AMC. Proteolytic activity of USP21 was observed by the increase of fluorescence emission at 460 nm with excitation at 360 nm for 30 min using a BioTek Synergy 2 plate reader (BioTek Instruments). Activity of USP21 was normalized to noninhibited USP21, and the percent activity was plotted versus the ubiquitin variant concentrations with a log scale. IC50 values were calculated by fitting a dose-response curve with a four-parameter logistic function.
10
Ubiquitin Cleavage Kinetics Assay
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All reactions were performed in Ub-AMC assay buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1 mM ATP, 5 mM MgCl2, 1 mM DTT, and 1 mg/mL ovalbumin [Sigma]), with a final reaction vol of 20 μL per assay in a 384-well plate (Corning). Ub-AMC cleavage was monitored by measuring fluorescence in real time for at least 30 min at 365 nm excitation and 460 nm emission with an EnVision plate reader (Perkin Elmer, ICCB Facility, HMS). Analysis was performed using Microsoft Excel and GraphPad PRISM, the initial kinetics observed within the linear range was used for plotting. For routine assays, Ub-AMC (Boston Biochem) was used at a concentration (0.5 to 1 μM) far below the KM, so that activity measurements were performed under kcat/KM conditions. All Ub-AMC assays were independently repeated and yielded consistent results.
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