ECM600 | uPA Activity Assay Kit

ECM600
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      Overview

      Replacement Information

      Key Spec Table

      Species ReactivityKey ApplicationsDetection Methods
      HACTColorimetric
      Description
      Catalogue NumberECM600
      Brand Family Chemicon®
      Trade Name
      • Chemicon
      DescriptionuPA Activity Assay Kit
      OverviewUrokinase-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.
      Materials Required but Not Delivered1. Single or Multichannel Pipette and disposable tips

      2. Microplate reader (405 nm)

      3. 37°C incubator

      4. Clean 96-well microplate for performing incubations.
      References
      Product Information
      Components
      • 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.
      Detection methodColorimetric
      Applications
      ApplicationThe uPA Activity Assay Kit provides a quick, efficient & sensitive system for evaluation of uPA activity & for screening of uPA inhibitors.
      Key Applications
      • Activity Assay
      Application NotesNotes 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.
      Biological Information
      Species Reactivity
      • Human
      Entrez Gene Number
      Entrez Gene SummaryThis gene encodes a serine protease involved in degradation of the extracellular matrix and possibly tumor cell migration and proliferation. A specific polymorphism in this gene may be associated with late-onset Alzheimer disease and also with decreased affinity for fibrin-binding. The protein encoded by this gene converts plasminogen to plasmin by specific cleavage of an Arg-Val bond in plasminogen. This gene's proprotein is cleaved at a Lys-Ile bond by plasmin to form a two-chain derivative in which a single disulfide bond connects the amino-terminal A-chain to the catalytically active, carboxy-terminal B-chain. This two-chain derivative is also called HMW-uPA (high molecular weight uPA). HMW-uPA can be further processed into LMW-uPA (low molecular weight uPA) by cleavage of chain A into a short chain A (A1) and an amino-terminal fragment. LMW-uPA is proteolytically active but does not bind to the uPA receptor.
      Gene Symbol
      • 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].
      UniProt Number
      UniProt SummaryFUNCTION: SwissProt: P00749 # Specifically cleave the zymogen plasminogen to form the active enzyme plasmin.
      SIZE: 431 amino acids; 48525 Da
      SUBUNIT: Found in high and low molecular mass forms. Each consists of two chains, A and B. The high molecular mass form contains a long chain A which is cleaved to yield a short chain A. Binds LRP1B; binding is followed by internalization and degradation. Interacts with MRC2. Interacts with PLAUR.
      SUBCELLULAR LOCATION: Secreted.
      TISSUE SPECIFICITY: Expressed in the prostate gland and prostate cancers.
      PTM: Phosphorylation of Ser-158 and Ser-323 abolishes proadhesive ability but does not interfere with receptor binding.
      SIMILARITY: SwissProt: P00749 ## Belongs to the peptidase S1 family. & Contains 1 EGF-like domain. & Contains 1 kringle domain. & Contains 1 peptidase S1 domain.
      Physicochemical Information
      Sensitivity
      • 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
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • 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.
      Storage and Shipping Information
      Storage ConditionsStore 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.
      Packaging Information
      Material Size1 kit
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      SDS

      Title

      Safety Data Sheet (SDS) 

      References

      Reference overviewPub Med ID
      Functional dissection of the PE domain responsible for translocation of PE_PGRS33 across the mycobacterial cell wall.
      Alessandro Cascioferro,Maria H Daleke,Marcello Ventura,Valentina Donà,Giovanni Delogu,Giorgio Palù,Wilbert Bitter,Riccardo Manganelli
      PloS one  6  2011

      Show Abstract Full Text Article
      22110736 22110736
      Regulation of STIM1 and SOCE by the ubiquitin-proteasome system (UPS).
      Jeffrey M Keil,Zhouxin Shen,Steven P Briggs,Gentry N Patrick
      PloS one  5  2010

      Show Abstract Full Text Article
      20976103 20976103
      The glutamate release inhibitor Riluzole decreases migration, invasion, and proliferation of melanoma cells.
      Maithao N Le,Joseph L-K Chan,Stephen A Rosenberg,Adam S Nabatian,Kim T Merrigan,Karine A Cohen-Solal,James S Goydos
      The Journal of investigative dermatology  130  2010

      Show Abstract
      20505744 20505744
      Qualitative and quantitative differences in the intensity of Fas-mediated intracellular signals determine life and death in T cells.
      Min-Jung Shin,Jae-Hyuck Shim,Jae-Young Lee,Wook-Jin Chae,Heung-Kyu Lee,Tomohiro Morio,Jun Han Park,Eun-Ju Chang,Sang-Kyou Lee
      International journal of hematology  92  2010

      Show Abstract
      20658220 20658220
      Molecular evolution of specific human antibody against MUC1 mucin results in improved recognition of the antigen on tumor cells.
      Jonas Persson,Malin Bäckström,Henrik Johansson,Karin Jirström,Gunnar C Hansson,Mats Ohlin
      Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine  30  2009

      Show Abstract
      19776674 19776674
      Rev and Rex proteins of human complex retroviruses function with the MMTV Rem-responsive element.
      Jennifer A Mertz,Mary M Lozano,Jaquelin P Dudley
      Retrovirology  6  2009

      Show Abstract Full Text Article
      19192308 19192308
      Dopamine D1 receptor modulates hippocampal representation plasticity to spatial novelty.
      Anh Hai Tran,Teruko Uwano,Tatsuo Kimura,Etsuro Hori,Motoya Katsuki,Hisao Nishijo,Taketoshi Ono
      The Journal of neuroscience : the official journal of the Society for Neuroscience  28  2008

      Show Abstract
      19074012 19074012
      Identification and biochemical characterization of unique secretory nucleases of the human enteric pathogen, Entamoeba histolytica.
      Glen C McGugan,Manju B Joshi,Dennis M Dwyer
      The Journal of biological chemistry  282  2007

      Show Abstract
      17766245 17766245
      Molecular analysis of PIP2 regulation of HERG and IKr.
      Jin-Song Bian,Anna Kagan,Thomas V McDonald
      American journal of physiology. Heart and circulatory physiology  287  2004

      Show Abstract
      15231497 15231497
      Identification of CC chemokine receptor 7 residues important for receptor activation.
      Thomas R Ott,Anil Pahuja,Sarah A Nickolls,David G Alleva,R Scott Struthers
      The Journal of biological chemistry  279  2004

      Show Abstract
      15284247 15284247

      Data Sheet

      Title
      uPA Activity Assay Kit - Data Sheet
      uPA Activity Assay Kit - Data Sheet