Tissue Factor (TF) Chromogenic Activity Assay Kit (Two Steps, Lipoprotein)
(1 reference)-
- 抗原
- Tissue Factor (TF) (Two Steps, Lipoprotein)
- 适用
- 人
- 检测方法
- Colorimetric
- 最低检测浓度
- < 1.5 pM
- 应用范围
- Activity Assay (AcA)
- 原理
- The AssaySense Human TF Chromogenic Activity Assay Kit is developed to determine human TF chromogenic activity in plasma, tissue, and cell culture supernatants
- 样品类型
- Plasma, Cell Culture Supernatant
- 特异性
- This assay recognizes both natural and recombinant human TF.
- 组件
- The activity assay kit contains sufficient reagents to perform 100 tests using microplate method. TF 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. Sample Diluent: 30 ml Assay Diluent: 20 ml rhTF Standard (lipoprotein): 1 vial recombinant human TF lipoprotein Human FVII: 1 vial Human Fx: 1 vial Fxa Substrate: 2 vials Storage Condition
- 试剂未包括
- 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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- 实验流程
- The assay measures the ability of lipoprotein TF/FVIIa to activate factor x (Fx) to factor xa. The amidolytic activity of the TF/FVIIa complex is quantitated by the amount of Fxa produced using a highly specific Fxa substrate releasing a yellow para-nitroaniline (pNA) chromophore. The change in absorbance of the pNA at 405 nm is directly proportional to the TF enzymatic activity.
- 试剂准备
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Standard Curve: Reconstitute the TF Standard with 1 ml of reagent grade water to generate a solution of 2 nM. 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 (2 nM) 1:4 with Sample Diluent to produce 500, 125, 31.25, 7.81, and 1.95 pM. Sample Diluent serves as the zero standard (0 pM). 2 Standard Point Dilution [TF] (pM) P1 1 part Standard (2 nM) + 3 part Sample Diluent 500.00 P2 1 part P1 + 3 part Sample Diluent 125.00 P3 1 part P2 + 3 part Sample Diluent 31.25 P4 1 part P3 + 3 part Sample Diluent 7.81 P5 1 part P4 + 3 part Sample Diluent 1.95 P6 Sample Diluent 0.00 FVII: Add 1.2 ml reagent grade water. Fx: Add 1.2 ml reagent grade water. Fxa Substrate: Add 1.1 ml reagent grade water.
- 样品收集
- Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes and assay. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: The cultured cells are lysed and solubilized with 15 mM octyl- -D-glucopyranoside at 370C for 15 minutes. Collect fresh cell lysates and assay. The samples can be stored at -20°C or below for up to 3 months Tissue: Extract tissue samples using 50 mM Tris-buffered saline (pH 8.0) with 1% Triton x- 100 and centrifuge at 14000 x g for 20 min. Collect the supernatant and measure the protein concentration. Dilute the tissue extract 1:4 into Sample Diluent and assay. Freeze the remaining extract at
- 实验流程
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Freshly prepare the desired volume of the Assay Mix by combining the following reagents according to the assay numbers (n).
- 结果分析
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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 The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.
- 限制
- 仅限研究用
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- 储存条件
- 4 °C/-20 °C
- 储存方法
- 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.
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: "Proof-of-concept study demonstrating the pathogenicity of affinity-purified IgG antibodies directed to domain I of β2-glycoprotein I in a mouse model of anti-phospholipid antibody-induced thrombosis." in: Rheumatology (Oxford, England), Vol. 54, Issue 4, pp. 722-7, (2015) (PubMed).
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: "Proof-of-concept study demonstrating the pathogenicity of affinity-purified IgG antibodies directed to domain I of β2-glycoprotein I in a mouse model of anti-phospholipid antibody-induced thrombosis." in: Rheumatology (Oxford, England), Vol. 54, Issue 4, pp. 722-7, (2015) (PubMed).
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- 抗原
- Tissue Factor (TF) (Two Steps, Lipoprotein)
- 背景
- The transmembrane protein Tissue factor (TF) is the physiologic trigger of coagulation in normal hemostasis. TF binds and allosterically activates factor VII (FVII). The TF-FVIIa complex cleaves factor Ix and x, leading to thrombin generation. TF markedly enhances the ability of FVIIa to cleave both macromolecule and small peptidyl substrates. Inducible expression of TF in a variety of pathological conditions, including gram-negative sepsis and acute coronary syndromes, is associated with life-threatening thrombosis. In sepsis, TF expression within the vasculature leads to disseminated intravascular coagulation. TF also plays important roles in vasculogenesis, metastasis, and tumor-associated angiogenesis.
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