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

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

Description

Urokinase Activity Assay Kit (Fluorometric), also known as the uPA Activity Assay Kit, u-Plasminogen Activator Activity Assay Kit, or PLAU Activity Assay Kit, is a kit designed to rapidly and sensitively detect urokinase activity in cells, blood, and other samples through fluorometry. It utilizes the catalytic action of urokinase on a substrate to produce a fluorescent substance, AMC, which can be measured.

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 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

• Buffer A for Enzyme Assay
• Assay Buffer
• uPA Standard (10,000IU/ml)
• Substrate (5X)

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. The activity of uPA is proportional to the fluorescence intensity. AMC has a maximum excitation wavelength of 350 nm and a maximum emission wavelength of 450 nm.

Features

• This kit offers high sensitivity, a wide linear range, and requires only a small sample volume. Compared to Urokinase Activity Assay Kit (Colorimetric), this kit has higher detection efficiency and sensitivity. After 60 minutes of incubation, it can detect as low as approximately 0.02 IU of uPA, with a good linear relationship within the range of 0.02-2.5 IU. The kit includes a urokinase standard solution (uPA Standard), allowing for the calculation of uPA activity in samples by setting up a standard curve. Typically, 1-10 μl of cell samples is sufficient for uPA activity detection in a 96-well plate. If the uPA activity in the sample is particularly low, Urokinase Activity Assay Kit (Colorimetric), which has more flexible detection times and can incubate overnight, can be used.

• This kit is highly flexible, fast, and widely applicable. The conditions for the Assay Buffer, incubation temperature, and reagent volumes have been optimized, making it suitable for both small sample detection and high-throughput screening in automated systems. It can be used to detect human serum and cell or tissue samples. The kit employs a one-step detection method, making it convenient, simple, and quick to use, completing the process in approximately 0.5-1 hour.

• The detection lysis buffer provided in this kit is versatile. The kit includes Buffer A for Enzyme Assay, which can directly lyse cell samples for detecting uPA enzyme activity. This buffer can also be used for detecting samples in other enzyme assay kits that use Buffer A for Enzyme Assay, making it highly versatile. Additionally, it can be used for protein concentration detection, SDS-PAGE, or Western blot detection of easily soluble proteins.

Storage

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

Precautions

• Due to the specificity of the substrate, this product can also detect tissue-type plasminogen activator (t-PA), thrombin, or proteases with trypsin-like activity. If the sample contains t-PA, thrombin, or trypsin-like proteases, it may interfere with the detection of urokinase activity. In such cases, set up a sample background control with a urokinase inhibitor during sample detection. The signal difference between the sample and the sample background control wells represents the signal value of AMC released by urokinase catalysis. The recommended urokinase inhibitor is GGACK (Urokinase Inhibitor).
• The Assay Buffer, Substrate (10X), and GGACK (10mM) should be completely thawed and equilibrated to room temperature before use to ensure accurate detection results. uPA Standard (10,000IU/ml) should be kept on ice during use, and all reagents should be stored according to the kit's requirements immediately after use.
• When preparing samples with the Buffer A for Enzyme Assay provided in this kit, and the sample amount is within the recommended range, it can ensure that the reaction system's pH value is within the appropriate range. If using a self-prepared lysis buffer, 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.
• 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.
• If serum or cell lysate samples are stored at 4ºC for uPA activity detection, the storage time should not exceed 2 days, otherwise, the accuracy of the detection results may be affected. Typically, cell lysate samples should be stored at -20ºC, and storage at -80ºC is preferable.
• For fluorescence microplate reader detection, use black or white plates suitable for fluorescence detection. 
• 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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