Product Datasheet

Ub-AMC (Ubiquitin-7-amino-4-methylcoumarin)

QW-001

Hefei Tsingwan Biotechnology Co., Ltd.

1Product Overview

Product Overview

Ub-AMC is a fluorogenic substrate in which the C-terminus of ubiquitin is covalently conjugated to the fluorescent reporter AMC (7-amino-4-methylcoumarin) via an isopeptide bond. It is the most widely used and highly cited classic tool reagent for deubiquitinase (DUB) activity assays.

Detection Principle

In the intact Ub-AMC molecule, AMC fluorescence is effectively quenched by the ubiquitin moiety. Upon DUB-mediated cleavage of the C-terminal isopeptide bond, free AMC is released, generating a dramatic fluorescence increase (Ex 360 nm / Em 460 nm). The rate of fluorescence increase is directly proportional to DUB enzyme activity and can be monitored in real time using a fluorescence plate reader or spectrofluorometer.

Applications

DUB enzyme kinetics (Km, kcat, Vmax)
High-throughput DUB inhibitor screening and IC50 determination
Total DUB activity quantification in cell lysates
Reaction condition optimization (pH, temperature, ionic strength)
Ubiquitin-proteasome pathway research

Product Highlights

HPLC purity ≥95% with excellent batch-to-batch consistency
Low background, high signal-to-noise ratio for trace enzyme detection
Every batch verified by MS and HPLC
Available in 1 mg and 5 mg sizes; custom bulk orders supported

Experimental Tips

Recommended final substrate concentration: 0.5–5 μM (adjust based on enzyme level). Use Tris-HCl (pH 7.4) with 1 mM DTT as reaction buffer. Initiate reaction by adding substrate and monitor fluorescence continuously for 30–60 min.

2Product Specifications

Cat. No.	QW-001
Molecular Formula	C₃₈₈H₆₃₆N₁₀₆O₁₁₉S
Molecular Weight	8,722 Da
Purity	≥95% (HPLC)
Specification	1mg / 5mg / 定制
Ex	360 nm
Em	460 nm
Storage	-80°C 避光保存

3Analytical Data

Mass Spectrometry Analysis

Reversed-phase high-performance liquid chromatography coupled with mass spectrometry (RP-HPLC-MS) was employed to analyze Ub-AMC. The measured molecular weight is 8,721.7 Da, in excellent agreement with the calculated theoretical value of 8,722 Da.

Figure 1　Mass Spectrum of Ub-AMC. The major peak corresponds to 8,721.7 Da, consistent with the theoretical MW of 8,722 Da.

Chromatographic Analysis (RP-HPLC)

Reversed-phase HPLC was performed to assess the purity of Ub-AMC. Chromatographic conditions: Welch® XB-C18 column (4.6×250 mm, 5 μm), flow rate 1.0 mL/min, detection wavelength 214 nm. Purity ≥ 95%.

Figure 2　RP-HPLC Chromatogram of Ub-AMC. Mobile phase A: 1% CH₃CN + 0.1% TFA; Mobile phase B: 1% water + 0.1% TFA. Gradient: 20% B → 70% B over 30 min.

Purity Analysis (SDS-PAGE)

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to verify the molecular weight and purity of Ub-AMC. The Coomassie Brilliant Blue R-250 staining result is shown below.

Figure 3　SDS-PAGE Analysis of Ub-AMC. A single, distinct band at approximately 8.7 kDa confirms ≥95% purity.

Fluorescence Hydrolysis Assay

50 μg Ub-AMC was dissolved in DMSO and diluted to 287 μL (20 μmol/L) in 50 mM Tris / 100 mM NaCl / 1 mM DTT / 1 mM EDTA (pH 7.5). 200 μL substrate was incubated with 0.2 μL UCHL3 (2.07 mg/mL) in a black 96-well plate; fluorescence monitored on Thermo Scientific Varioskan LUX (Ex 360 nm / Em 460 nm).

Figure 4　Fluorescence Hydrolysis Curve of Ub-AMC. Upon DUB-mediated hydrolysis, the released AMC fluorescence intensity increases progressively over time.