uPA Activity Assay Kit; 1 kit

uPA Activity Assay Kit

Chemicon (Millipore)

Urokinase-type Plasminogen Activator (uPA) is a 52 kDa serine protease which has been implicated in a number of physiological and pathological processes, including tissue remodeling1, angiogenesis2, fibrinolysis and tumor spread. When bound to its cell surface receptor, uPA is converted from the single chain pro-form uPA to the active 2-chain HMW-uPA. uPA has been shown to play a role in basement membrane degradation, via a cascade involving activation of plasminogen and the matrix metalloproteinases3. Inhibitors of uPA have been shown to slow primary tumor growth and metastasis4-7.

The CHEMICON uPA Activity Assay Kit provides a quick, efficient and sensitive system for evaluation of uPA activity and for screening of uPA inhibitors. The assay is colorimetric and does not require radioactivity or fluorescence equipment. The assay is sensitive over a range of 0.05-50 units of uPA activity.

Test Principle:

The CHEMICON uPA Activity Assay Kit utilizes a chromogenic substrate, which is cleaved by active uPA. Addition of this substrate to a uPA-containing sample results in a colored product, detectable by its Optical Density at 405nm (OD405).

Application:

The CHEMICON uPA Assay Kit is ideal for measurement of uPA activity in purified preparations and cell culture, as well as in serum where pathological conditions such as sepsis exist. The assay is also useful for screening inhibitors of the enzymatic activity of uPA.

Each CHEMICON uPA Activity Assay Kit contains sufficient reagents for the evaluation of 96 samples, including uPA from human urine as a positive control. Duplicate or triplicate samples are suggested.

The CHEMICON uPA Activity Assay Kit is intended for research use only; not for diagnostic or therapeutic applications.

Activity Assay

Notes on uPA activity assay sample preparation.



Plasma: Perform venepuncture of the cubial vein, with minimal stasis after a rest period of at least 10min. Discard the first few milliliters of blood. Collect nine volumes of blood in one volume of cold sodium citrate, 0.11 mol/L, in a polycarbonate tube, mix gently and place on ice. Subsequently, prepare platelet-poor plasma by centrifugation in a refrigerated centrifuge (30 min, 2000g). Snap-freeze plasma samples of 0.3 mL to 0.6 mL and store at -60°C. Before use, thaw plasma rapidly in a water bath at 37°C and put on ice.



Tissue: There are numerous publications for the extraction of uPA from tissues. A suitable buffer for extraction of uPA from the membrane fraction requires Triton X100. A non-detergent extract (also called cytosolic fraction) does not require Triton, however the membrane fraction is removed by ultracentrifugation, so uPA activity associated with the membrane fraction is bypassed by measuring only the cytosols. Below are some references that describe extraction procedures for uPA.

Cancer Res. (1987). 47(17):4654-4657.

J. Bone Miner Res. (1991). 6(10):1081-1090.

Cancer Res. (1994). 54(10):2527-2530.

Br. J. Cancer (1996). 74(8):1168-1174.



Cell culture: Isolate cell culture supernatant and use directly.

Human

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

• Apoptosis Assays
• Cancer Kits
• Enzyme Activity Assays

• uPA Positive Control: (Part No. 90058) One lyophilized vial, 1000 units, of uPA from human urine.
• Chromogenic Substrate: (Part No. 90057) One 5 mg bottle of Tripeptide with pNA group.
• Assay Buffer, 10X: (Part No. 90091) One 5 mL bottle.

Positive Control: Specific activity: =100,000 units/mg protein. One unit is defined as the amount of enzyme equal to an international standard tested by the fibrinolytic method of Johnson, et al. {Johnson, A.J., et al. 1969. Thromb. Diath. Haemorrh. 21, 259.}

~10 μg of standard is supplied in ECM600

Store kit materials at -20°C for up to 6 months. After reconstitution, store uPA positive control at -70°C and substrate at 2 - 8°C.

Colorimetric

• PLAU
• UPA
• URK
• u-PA
• uPA
• ATF
• EC 3.4.21.73 [Contains: Urokinase-type plasminogen activator long chain A
• Urokinase-type plasminogen activator short chain A
• Urokinase-type plasminogen activator chain B].

1. Single or Multichannel Pipette and disposable tips

2. Microplate reader (405 nm)

3. 37°C incubator

4. Clean 96-well microplate for performing incubations.