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Cat. No.: UISKF-100 (for 100T)

Cat. No.: UISKF-500 (for 500T)

Description

Urokinase Inhibitor Screening Kit (Fluorometric), also known as the uPA Inhibitor Screening Kit, u-Plasminogen Activator Inhibitor Screening Kit, or PLAU Inhibitor Screening Kit, is a kit designed to rapidly and sensitively screen or detect urokinase inhibitors (uPA inhibitors) through fluorometry. It uses urokinase to catalyze the substrate, generating a fluorescent substance, AMC, which can be measured. This kit is suitable for both small sample detection and high-throughput screening in automated systems.

In the human body, the most important plasminogen activators are tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), both of which are secreted serine proteases. t-PA is primarily expressed and secreted by vascular endothelial cells and smooth muscle cells, continuously released into the bloodstream. u-PA is expressed by macrophages in atherosclerotic vessels and other cells.

Urokinase-type plasminogen activator (uPA or u-PA), also known as urokinase, was first isolated from human urine and is also found in the blood and extracellular matrix of many tissues. It plays a key role in ECM remodeling, cell migration, inflammation, and cancer. uPA's main substrate is plasminogen, which it catalyzes into active plasmin, triggering a proteolytic cascade that participates in thrombolysis or ECM degradation. This cascade is linked to vascular diseases and cancer progression. uPA consists of an EGF-like domain, Kringle domain, and serine protease domain. It is secreted as an inactive zymogen and activated by proteolytic cleavage, resulting in active uPA—a disulfide-linked dimer composed of an N-terminal A-chain (EGF-like and Kringle domains) and a C-terminal B-chain (serine protease).

Components

• Assay Buffer
• uPA
• Substrate (5X)
• GGACK (10mM)

Principle

The uPA substrate provided in this kit carries the AMC group, which urokinase cleaves through proteolysis, releasing the fluorescent substance AMC. The fluorescence of AMC can be easily measured to detect uPA enzyme activity. If a uPA inhibitor is added to the reaction system, the production of the fluorescent product AMC will be inhibited. The fluorescence intensity is inversely proportional to the inhibitory effect of the inhibitor, allowing for the detection of the inhibitor's effect. AMC has a maximum excitation wavelength of 350 nm and a maximum emission wavelength of 450 nm.

Features

• This kit offers high sensitivity and strong specificity. It includes urokinase (uPA), a urokinase substrate, and a positive control urokinase inhibitor, GGACK. The usage amounts of urokinase and the substrate have been optimized to detect both low and high IC50 inhibitors. The GGACK provided in this kit is an irreversible urokinase inhibitor.
• This kit has strong compatibility. Common solvents such as DMSO, anhydrous ethanol, and glycerol have minimal impact on the detection results of this kit. Experimental results show that at a concentration of 20% in the reaction system, the signal reduction after 1 hour of incubation is less than 10% for DMSO, 8% for anhydrous ethanol, and 6% for glycerol. For detergents like Triton X-100, at a concentration of 1.5%, the signal reduction after 1 hour of incubation does not exceed 15%. By setting up a solvent control during detection, the influence of solvents on the detection system can be effectively eliminated.

Storage

Store at -20ºC, valid for one year. The Substrate (5X) should be stored protected from light, and both the Substrate (5X) and GGACK (10mM) should avoid repeated freeze-thaw cycles.

Precautions

• The Assay Buffer, Substrate (5X), and GGACK (10mM) should be completely thawed and equilibrated to room temperature before use to ensure accurate detection results. uPA should be kept on ice during use, and all reagents should be stored according to the kit's requirements immediately after use.
• For small-volume reagents in this kit, it is recommended to centrifuge briefly to settle the liquid at the bottom of the tube before use. Thawed reagents must be fully melted and mixed well before use.
• For microplate reader detection, use transparent plates suitable for colorimetric detection.
• Ensure that the pH value of the reaction system is between 7.5-8 after adding the sample, or that the sample's pH is between 7.5-8, otherwise it may affect the signal value and stability of the detection results.
• The solvent of the inhibitor samples to be tested may interfere with detection. It is recommended to use the Assay Buffer provided in this kit as the solvent for preparing and diluting samples. If the samples must be prepared and diluted with other reagents, conduct preliminary testing and add an equal volume of solvent to the control wells to eliminate interference.
• This product is intended for scientific research use only by professionals and must not be used for clinical diagnosis or treatment, food or drugs, or stored in a regular household.
• For your safety and health, wear a lab coat and disposable gloves while handling.

Related:

• Urokinase Activity Assay Kit (Colorimetric)
• Urokinase Inhibitor Screening Kit (Colorimetric)
• Urokinase Activity Assay Kit (Fluorometric)
• Urokinase Inhibitor Screening Kit (Fluorometric)

Only for research and not intended for treatment of humans or animals

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