Human Thrombin Chromogenic Activity Assay Kit

Catalog No. ABIN5564624

Product Details

Target: alpha-Thrombin

Reactivity: Human

Detection Method: Colorimetric

Detection Range: 0.05-1.6 U/mL

Minimum Detection Limit: 0.05 U/mL

Application: Activity Assay (AcA)

Purpose: The AssaySense Human Alpha Thrombin Chromogenic Activity Kit is developed to determine human thrombin activity in plasma, serum, and cell culture samples. The amidolytic activity of thrombin is quantitated using a highly specific thrombin substrate releasing a (pNA) chromophore. The change in absorbance of the pNA at 405 nm is directly proportional to the thrombin enzymatic activity.

Brand: AssaySense

Sample Type: Cell Culture Cells, Plasma, Serum

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 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. 2 EIA Diluent Concentrate (10x): A 10-fold buffered protein base (20 ml). Human Thrombin Standard: 1 vial, lyophilized (0.48 AU) Thrombin Substrate: 2 vials, lyophilized

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

Comment:

Chromogenic activity kits are developed to determine the activity of specific proteins in plasma, serum, and/or other body fluids. Activity is measured using either direct or indirect amidolytic assay formats or via coupling with immunofunctional formats. The amidolytic activity of the protein of interest is quantified using a highly specific substrate which releases a yellow para-nitroaniline (pNA) chromophore. The change in absorbance of the pNA at 405 nm is proportional to enzymatic activity of the targeted protein.

Protocol:

• Step 1. Add 10 μL of Standard or Sample per well. Add 90 μL of AssayMix per well. Read at 405 nm at 0 minutes. Incubate at 37 °C.
• Step 2. Read every 30 minutes for 2 hours at 405 nm.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. 3 EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 1:10 with reagent grade water. Store for up to 30 days at 2-8 °C.

Assay Procedure:

Add 10 l of Thrombin Standard or sample per well. AssayMix: Freshly prepare the desired volume of the AssayMix by combining the following reagents according to the assay numbers (n). Assay Mix

Calculation of Results:

• 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 (OD) 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.

Restrictions: For Research Use only

Handling

Handling Advice: This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents 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 insert. However, the user should determine the optimal dilution factor. The kit should not be used beyond the expiration date.

Storage: 4 °C,-20 °C

Storage Comment: Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store Standard and Thrombin Substrate at -20°C. Store Microplate and EIA Diluent (10x) at 2-8°C. Unused microplate wells may be returned and resealed. Diluted EIA Diluent (1x) may be stored for up to 30 days at 2-8°C.

Target Details

Target: alpha-Thrombin

Alternative Name: Thrombin (alpha-Thrombin Products)

Synonyms: PT Kit, RPRGL2 Kit, THPH1 Kit, coagulation factor II, thrombin Kit, F2 Kit

Background: Thrombin (activated Factor II [IIa]) is a coagulation protein that has many effects in the coagulation cascade. Thrombin is a serine protease (EC 3.4.21.5) that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions (1). Thrombin is in the form of alpha-thrombin that is the immediate end product of prothrombin activation: two further thrombin products can be identified, beta- and gamma- thrombin. These are degraded forms that may arise from autodigestion of a thrombin preparation (2, 3).

Gene ID: 2147

UniProt: P00735