PAI-1 Chromogenic Activity Assay Kit

Catalog No. ABIN612647

Product Details

Target: PAI1 (SERPINE1) (Plasminogen Activator Inhibitor 1 (SERPINE1))

Reactivity: Human

Detection Method: Colorimetric

Minimum Detection Limit: 5 AU/mL

Application: Activity Assay (AcA)

Purpose: The AngioSense Human PAI-1 Chromogenic Activity Assay Kit is developed to determine human PAI-1 activity in cell culture supernatants

Sample Type: Cell Culture Supernatant

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 tPA Standard: 2 vials human tPA (88 IU) Human Plasminogen: 1 vial Plasmin Substrate: 2 vials Storage Condition

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)

Application Details

Protocol: A fixed amount of tPA is added in excess to undiluted sample, which allows PAI-1 and tPA to form an inactive complex. The assay measures plasminogen activation by residual tPA in coupled 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 absorbance of the pNA at 405 nm is inversely proportional to the PAI-1 enzymatic activity.

Reagent Preparation:

Standard Curve: Reconstitute the tPA Standard (88 IU) with 2.2 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) with desired volume of Assay Diluent as below to produce 30, 20, and 10 IU/ml. Assay Diluent serves as the zero standard (0 IU/ml). Standard Dilution [tPA] (IU/ml) [PAI-1] (AU/ml) Point P1 Standard (40 IU/ml) 40.00 0.00 75 µl P1 + 25 µl Assay Diluent P2 30.00 10.00 50 µl P1 + 50 µl Assay Diluent P3 20.00 20.00 25 µl P1 + 75 µl Assay Diluent P4 10.00 30.00 P5 Assay Diluent 0.00 40.00 Note: One arbitrary unit (AU) of inhibitor is defined as the amount that inhibits one IU of tPA/ml under the testing conditions. Plasminogen: Add 1.2 ml reagent grade water. Plasmin Substrate: Add 1.1 ml reagent grade water. 2

Sample Collection: Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris. Collect supernatants and mix 20 µl samples with 20 µl tPA (40 IU/ml) for 10 minutes at room temperature prior to the assay.

Assay Procedure:

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

Calculation of Results:

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. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

Assay Precision: Intra-assay and inter-assay coefficients of variation were 5.1 % and 8.8% respectively.

Restrictions: For Research Use only

Handling

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 Details

Target: PAI1 (SERPINE1) (Plasminogen Activator Inhibitor 1 (SERPINE1))

Alternative Name: PAI-1 (SERPINE1 Products)

Synonyms: PAI Kit, PAI-1 Kit, PAI1 Kit, PLANH1 Kit, SERPINE1 Kit, si:ch211-138a11.1 Kit, Planh1 Kit, PAI1A Kit, Pai1 Kit, Pai1aa Kit, Planh Kit, RATPAI1A Kit, serpin family E member 1 Kit, serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 L homeolog Kit, serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 Kit, serine (or cysteine) peptidase inhibitor, clade E, member 1 Kit, SERPINE1 Kit, serpine1.L Kit, serpine1 Kit, Serpine1 Kit

Background: Type I plasminogen activator inhibitor (PAI-1) is a 50 kDa serpin family member that inhibits tissue- and urokinase-type plaminogen activators (t-PA, u-PA). This protein appears to be an important regulator of plasminogen activation by t-PA and extracellular proteolysis by u-PA. The plasminogen activator proteolytic enzyme systems are important not only for fibrinolysis but also for extracellular matrix remodeling, and have been implicated in a number of normal and pathological processes including angiogensis, ovulation and embryogenesis, thrombotic and hemorrhagic disorders, connective tissue diseases, neoplasm and sepsis. PAI-1 is a prognosticator in breast cancer, gastric cancer, various forms of lung cancer and cervical cancer.