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PLG Kit ELISA

PLG Reactivité: Boeuf (Vache) Colorimetric Competition ELISA Plasma, Serum
N° du produit ABIN1440254
  • Antigène Voir toutes PLG Kits ELISA
    PLG (Plasminogen (PLG))
    Reactivité
    • 6
    • 4
    • 4
    • 4
    • 3
    • 3
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    Boeuf (Vache)
    Méthode de détection
    Colorimetric
    Type de méthode
    Competition ELISA
    Seuil minimal de détection
    ~0.3 µg/mL
    Application
    ELISA
    Fonction
    The AssayMax Bovine Plasminogen ELISA kit is designed for detection of bovine plasminogen in plasma, serum, and cell culture supernatants. This assay employs a quantitative competitive enzyme immunoassay technique that measures Plasminogen in less than 3 hours. A polyclonal antibody specific for Plasminogen has been pre-coated onto a 96-well microplate with removable strips. Plasminogen in standards and samples is competed by a biotinylated Plasminogen sandwiched by the immobilized antibody and streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.
    Marque
    AssayMax
    Type d'échantillon
    Serum, Plasma
    Analytical Method
    Quantitative
    Réactivité croisée (Details)
    Cross-Reactivity: Canine 2%, Monkey 2%, Rat 4%, Human 4%, Swine 4%, Rabbit 0.5%, Mouse 2%.
    This assay recognizes both natural and recombinant bovine plasminogen.
    Attributs du produit
    Standard Added Value: 0.6 - 6 µg/mL
    Ingrédients
    Plasminogen Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against bovine plasminogen. 2
    Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay.
    Plasminogen Standard: Bovine plasminogen in a buffered protein base (24 µg, lyophilized).
    Biotinylated Plasminogen: 1 vial, lyophilized.
    EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 mL).
    Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 mL).
    Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 µL).
    Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 mL).
    Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 mL).
    Matériel non inclus
    Microplate reader capable of measuring absorbance at 450 nm
    Pipettes (1-20 µL, 20-200 µL, 200-1000 µL and multiple channel)
    Deionized or distilled reagent grade water
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  • Durée du test
    < 3 h
    Plaque
    Pre-coated
    Préparation des réactifs

    Freshly dilute all reagents and bring all reagents to room temperature before use.
    EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C.
    Standard Curve: Reconstitute the 24 µg of bovine Plasminogen Standard with 1 mL of EIA Diluent to produce 24 µg/mL of solution. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (24 µg/mL)1:2 with equal volume of EIA Diluent to produce 12, 6, 3, 1.5, 0.75, and 0.375 3 µg/mL of solutions. EIA Diluent serves as the zero standard (0 µg/mL). Any remaining solution should be frozen at -20°C.
    Biotinylated Plasminogen (2x): Dilute Biotinylated Plasminogen with 4 mL EIA Diluent to produce a 2-fold stock solution. Allow the biotin to sit for 10 minutes with gentle agitation prior to making dilutions. The stock solution should be further diluted 1:2 with EIA Diluent. Any remaining solution should be frozen at -20°C.
    Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 1:20 with reagent grade water.
    SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with EIA Diluent. Any remaining solution should be frozen at -20°C.

    Préparation de l'échantillon

    Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Dilute plasma 1:80 into EIA Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. (EDTA or Heparin can also be used as anticoagulant.)
    Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and assay. Dilute serum 1:80 into EIA Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze- thaw cycles.

    Procédure de l'essai

    Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-30°C).
    Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator.
    Add 25 µL of standard and/or sample per well, and immediately add 25 µL of Biotinylated Plasminogen to each well (on top of the standard or sample). Cover wells with a sealing tape and incubate for two hours at room temperature. Start the timer after the last sample addition.
    Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid.
    Add 50 µL of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance.
    Wash the microplate as described above.
    Add 50 µL of Chromogen Substrate per well and incubate for about 7 minutes or until the optimal blue color density develops. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip.
    Add 50 µL of Stop Solution to each well. The color will change from blue to yellow.
    Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.

    Calcul des résultats

    Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
    To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit.
    Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor.

    Précision du teste
    Intra-assay and inter-assay coefficients of variation were 4.7 % and 7.1 % respectively.
    Restrictions
    For Research Use only
  • Conseil sur la manipulation
    Prepare all reagents (working diluent buffer, wash buffer, standards, biotinylated-protein, and SP conjugate) as instructed, prior to running the assay.
    Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this protocol. However, the user should determine the optimal dilution factor.
    Spin down the SP conjugate vial before opening and using contents.
    The kit should not be used beyond the expiration date.
    The Stop Solution is an acid solution.
    Stock
    4 °C/-20 °C
    Stockage commentaire
    Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date.
    Store SP Conjugate at -20°C
    Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C
    Opened unused microplate wells may be returned to the foil pouch with the desiccant packs. Reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.
    Diluent (1x) may be stored for up to 1 month at 2-8°C.
    Store Standard and Biotinylated Protein at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.
  • Antigène Voir toutes PLG Kits ELISA
    PLG (Plasminogen (PLG))
    Autre désignation
    Plasminogen (PLG Produits)
    Synonymes
    wu:fb70e09 Kit ELISA, PLG Kit ELISA, LPA Kit ELISA, plg Kit ELISA, Ab1-346 Kit ELISA, AI649309 Kit ELISA, Pg Kit ELISA, plasminogen Kit ELISA, plg Kit ELISA, PLG Kit ELISA, Plg Kit ELISA
    Sujet
    Plasminogen is a single chain glycoprotein zymogen that is synthesized in the liver and circulated in plasma with a molecular weight of 90 kDa. The N-terminal portion of the molecule is made up of five kringle domains that bind to fibrin. The native molecule has an amino-terminal glutamic acid, known as glu-plasminogen, but this can undergo proteolytic cleavage by plasmin to lys-plasminogen. The inactive proenzyme plasminogen is converted to the active enzyme plasmin that ultimately digests fibrin. Tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA) catalyzes the activation of plasminogen, while plasminogen activator inhibitors (PAIs) inhibits the activation. The plasminogen system plays a role in macrophage recruitment, arterial stenosis, atherosclerosis, aneurysm formation, skin and corneal wound healing, glomerulonephritis, and neovascularization.
    Pathways
    Système du Complément, Lipid Metabolism
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