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

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

Description

Urokinase Activity Assay Kit (Colorimetric), 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 tissues, cells, blood, and other samples through colorimetry. It utilizes the catalytic action of urokinase on a substrate to produce a colored substance, pNA, 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 pNA group, which urokinase cleaves through proteolysis, releasing pNA. The absorbance of pNA can be easily measured to detect uPA enzyme activity, which is proportional to the absorbance detected. pNA has a strong absorption around 405 nm.

Features

• This kit offers high sensitivity, a wide linear range, and requires only a small sample volume. Compared to Urokinase Activity Assay Kit (Fluorometric), this kit provides more flexible detection times and a broader linear range. After 24 hours of incubation, this kit can detect as low as approximately 0.01 IU of uPA; after 60 minutes of incubation, it can detect as low as approximately 0.15 IU of uPA, with a good linear relationship within the range of 0-40 IU. The kit includes a urokinase standard solution (uPA Standard) for setting up a standard curve to calculate uPA activity in samples. Typically, 1-10 μl of sample is sufficient for uPA activity detection in a 96-well plate. If the uPA activity in the sample is low and ideal results are desired within 60 minutes, Urokinase Activity Assay Kit (Fluorometric), which has higher sensitivity for the same incubation time, can be used.
• This kit is flexible and widely applicable. The detection system has been optimized to accommodate both low and high uPA content samples, and it can be used to detect human serum and cell samples. The kit employs a one-step detection method, making it easy, simple, and quick to use.
• The detection lysis buffer provided in this kit has certain versatility. 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 pNA released by urokinase catalysis. The recommended urokinase inhibitor is GGACK (Urokinase Inhibitor).
• The Buffer A for Enzyme Assay, Assay Buffer, and Substrate (5X) should be completely thawed and equilibrated to room temperature before use to ensure accurate detection results. The uPA Standard (10,000IU/ml) should be kept on ice during use and 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 microplate reader detection, use transparent plates suitable for colorimetric 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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