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uPA Chromogenic Activity Assay Kit (Direct Assay)

AcA Reactivity: Human Colorimetric Plasma
Catalog No. ABIN612650
  • Target See all PLAU Kits
    PLAU (Plasminogen Activator, Urokinase (PLAU))
    Reactivity
    • 9
    • 4
    • 3
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    Human
    Detection Method
    Colorimetric
    Minimum Detection Limit
    0.1 IU/mL
    Application
    Activity Assay (AcA)
    Purpose
    The AngioSense Human uPA Chromogenic Activity Assay Kit is developed to determine human uPA activity in plasma and cell culture supernatants
    Sample Type
    Plasma
    Cross-Reactivity (Details)
    No significant cross-reactivity or interference was observed.
    Components
    The activity assay kit contains sufficient reagents to perform 100 tests using microplate method. Microplate: one 96-well polystyrene microplate (12 strips of 8 wells) Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Assay Diluent: 30 ml uPA Standard: 1 vial human high molecular weight uPA (1200 IU) uPA Substrate: lyophilized substrate (2 tubes)
    Material not included
    Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water Incubator (37 °C)
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  • Sample Volume
    20 μL
    Protocol
    The amidolytic activity of uPA is quantitated using a highly specific uPA substrate releasing a yellow para-nitroaniline (pNA) chromophore. The change in absorbance of the pNA at 405 nm is directly proportional to the uPA enzymatic activity.
    Reagent Preparation

    Substrate: Add 1.1 ml reagent grade water. Standard Curve: Reconstitute the uPA Standard with 3 ml of Assay Diluent to generate a stock solution of 400 IU/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. For high level of uPA activity samples, prepare triplicate standard points by serially diluting the standard solution (400 IU/ml) twofold with equal volume of Assay Diluent to produce 200, 100, 50, 25, 12.5 and 6.25 IU/ml. For low level of uPA activity samples, dilute the stock solution (400 IU/ml) 1:32 with Assay Diluent to yield a solution of 12.5 IU/ml. Prepare triplicate standard points by serially diluting the standard solution (12.5 IU/ml) twofold with equal volume of Assay Diluent to produce 6.250, 3.125, 1.563, 0.781, and 0.391 IU/ml. Assay Diluent serves as the zero standard (0 IU/ml). 2 Standard curve for high level of uPA activity samples: Standard Dilution [uPA] (IU/ml) Point P1 1 part Standard (200 IU/ml) 200.00 P2 1 part P1 + 1 part Assay Diluent 100.00 P3 1 part P2 + 1 part Assay Diluent 50.00 P4 1 part P3 + 1 part Assay Diluent 25.00 P5 1 part P4 + 1 part Assay Diluent 12.50 P6 1 part P5 + 1 part Assay Diluent 6.25 P7 Assay Diluent 0.00 Standard curve for low level of uPA activity samples: Standard Dilution [uPA] (IU/ml) Point P1 1 part Standard (12.5 IU/ml) 12.500 P2 1 part P1 + 1 part Assay Diluent 6.250 P3 1 part P2 + 1 part Assay Diluent 3.125 P4 1 part P3 + 1 part Assay Diluent 1.562 P5 1 part P4 + 1 part Assay Diluent 0.781 P6 1 part P5 + 1 part Assay Diluent 0.391 P7 Assay Diluent 0.000

    Sample Collection
    Plasma: Collect plasma using one-tenth volume of 0.11 M sodium citrate as an anticoagulant. Centrifuge samples at 2,000 x g for 10 minutes and assay.
    Assay Procedure

    Add 50 µL of the Assay Diluent to each well of the 96-well plate. Add 30 µL of uPA Standards or testing samples per well and mix gently. Add 20 µL of uPA Substrate to each well and mix gently. Seal the plate with a sealing tape. Incubate the plate at 370C in a humid incubator to avoid drying the plate. For HIGH uPA activity samples, read the absorbances at 405 nm periodically every 5 minutes up to 30 minutes. For LOW uPA activity samples, start to read the absorbances at 405 nm from 2 hours up to 6 hours. 50 µL Assay Diluent 30 µL uPA or Samples 20 µL uPA Substrate High uPA activyt Samples: 370C, read the absorbances at 405 nm every 5 minutes for 30 minutes Low uPA Activity samples: 370C, read the absorbances at 405 nm every hour from 2 hours to 6 hours

    Calculation of Results

    Calculate the mean value of the triplicate for each standard and sample. To generate a Standard Curve from the initial reaction time, plot the graph using the standard concentrations on the x-axis and the corresponding mean 405 nm absorbance or change in absorbance per minute (A/min) on the y-axis. The best-fit line can be determined by regression analysis of the linear portion of the curve. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

    Restrictions
    For Research Use only
  • Storage
    4 °C/-20 °C
    Storage Comment
    Store unopened kit at 2-8°C up to expiration date. Opened reagents may be stored for up to 1 month at 2-8°C. Store reconstituted standard and reagents at -20°C or below.
  • Target
    PLAU (Plasminogen Activator, Urokinase (PLAU))
    Alternative Name
    uPA (PLAU Products)
    Synonyms
    PLAU Kit, u-PA Kit, ATF Kit, BDPLT5 Kit, QPD Kit, UPA Kit, URK Kit, uPA Kit, UPAM Kit, plasminogen activator, urokinase Kit, urokinase-type plasminogen activator Kit, PLAU Kit, CpipJ_CPIJ002131 Kit, CpipJ_CPIJ006543 Kit, CpipJ_CPIJ013396 Kit, CpipJ_CPIJ013623 Kit, Plau Kit
    Background
    Urokinase-type plasminogen activator (uPA) is a highly restricted serine protease that converts the zymogen plasminogen to active plasmin, a broad-spectrum serine proteinase capable of degrading most of the major protein components of the extracellular matrix. Binding of uPA to its receptor and subsequent uPA mediated pericellular proteolysis are involved in many process including cell migration and tissue remodeling in angiogeenesis, atherogenesis, tumor cell metastasis, and ovulation. High level of uPA is a poor prognostic marker for aggressive breast cancer, aggressive prostate cancer, bladder cancer and gastric cancer.
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