tPA Chromogenic Activity Assay Kit

Produktnummer ABIN612648

Kurzübersicht für tPA Chromogenic Activity Assay Kit (ABIN612648)

Target: PLAT (Plasminogen Activator, Tissue (PLAT))

Reaktivität: Human

Nachweismethode: Colorimetric

Applikation: Activity Assay (AcA)

Proben: Plasma

Produktdetails

Untere Nachweisgrenze: 0.08 IU/mL

Verwendungszweck: The AssaySense Human tPA Chromogenic Activity Assay Kit is developed to determine human tPA activity in cell culture supernatants

Kreuzreaktivität (Details): No significant cross-reactivity or interference was observed.

Bestandteile: 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 tPA Standard: 1 vial human tPA (48 IU) Human Plasminogen: 1 vial Plasmin Substrate: 2 vials Storage Condition

Benötigtes Material: 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)

Anwendungsinformationen

Protokoll: The assay measures the ability of tPA to activate the plasminogen to plasmin in coupled or indirect assays that contain tPA, plasminogen, and a plsmin-specific synthetic substrate. The amount of plasmin produced is quantitated using a highly specific plasmin substrate releasing a yellow para-nitroaniline (pNA) chromophore. The change in absorbance of the pNA in the reaction solution at 405 nm is directly proportional to the tPA enzymatic activity.

Aufbereitung der Reagenzien:

Plasminogen: Add 1.2 ml reagent grade water. Plasmin Substrate: Add 1.1 ml reagent grade water. Standard Curve: Reconstitute the tPA Standard with 1.2 ml of reagent grade water to generate a solution of 40 IU/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare triplicate standard points by serially diluting the standard solution (40 IU/ml) 1:4 with Assay Diluent to produce 10, 2.5, 0.625, 0.156, and 0.039 IU/ml. 2 Standard Point Dilution [tPA] (IU/ml) P1 1 part Standard (40 IU/ml) 40.000 P2 1 part P1 + 3 part Assay Diluent 10.000 P3 1 part P2 + 3 part Assay Diluent 2.500 P4 1 part P3 + 3 part Assay Diluent 0.625 P5 1 part P4 + 3 part Assay Diluent 0.156 P6 1 part P5 + 3 part Assay Diluent 0.039 P7 Assay Diluent 0.000

Probennahme: Plasma: Collect plasma using one-tenth volume of acidified 0.5 M sodium citrate (pH 4.0) as an anticoagulant to prevent tPA-PAI complex formation. Centrifuge samples at 3000 x g for 15 minutes.

Testdurchführung:

Assay Mix: Freshly prepare the desired volume of the Assay Mix by combining the following reagents according to the assay numbers (n).

Ergebnisberechnung:

Calculate the mean value of the duplicate or 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. 3 Standard Curve 1) The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: 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.

Referenzen

McGuire, Bennett, Lansley, Popowicz, Varano della Vergiliana, Wong, Lee, Chakera: "Preclinical assessment of adjunctive tPA and DNase for peritoneal dialysis associated peritonitis." in: PLoS ONE, Vol. 10, Issue 3, pp. e0119238, (2015) (PubMed).

Kim, Park, Han, Kim, Lee, Yoon Park, Kang: "Phloretin suppresses thrombin-mediated leukocyte-platelet-endothelial interactions." in: Molecular nutrition & food research, Vol. 58, Issue 4, pp. 698-708, (2014) (PubMed).

Antigendetails

Target: PLAT (Plasminogen Activator, Tissue (PLAT))

Andere Bezeichnung: tPA

Hintergrund: Tissue-type plasminogen activator (tPA) is a 68 kDa serine protease that converts the zymogen plasminogen into the active serine protease plasmin which digests fibrin and induce the dissolution of fibrin clots. tPA is synthesized by endothelial cells in normal blood vessels and displays relatively high affinity for fibrin, suggesting that it functions predominately in physiological thrombolysis in vivo. High level of tPA is a good prognostic marker for breast cancer. tPA may minimize the formation of metastasis by preventing tumor cell adherence at sites of trauma. On the other hand, gastrointestinal cancer is accompanied by a decrease in tPA.