Recombinant Human t-Plasminogen Activator/tPA Protein, CF Summary
Met1-Pro562, with a C-terminal 6-His tag
Single-chain proform was expressed, purified, activated with thermolysin to a 2-chain protease and further purified.
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in MES, NaCl and CaCl2.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 50 mM Tris, 0.01% Tween-20, pH 8.5
- Recombinant Human t‑Plasminogen Activator/tPA (rhPLAT) (Catalog # 7449-SE)
- Fluorogenic Peptide Substrate Z-Gly-Gly-Arg-AMC (Bachem, Catalog # I-1140), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhPLAT to 2 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 µL of the 2 ng/µL rhPLAT into a black well plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate without any rhPLAT.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A9891).
- rhPLAT: 0.1 µg
- Substrate: 100 µM
Recombinant Human t-Plasminogen Activator (Catalog # 7449-SE) is measured by its ability to cleave a peptide substrate, N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC).
Background: t-Plasminogen Activator/tPA
PLAT, also known as tissue-type plasminogen activator (tPA), is a secreted serine protease synthesized by endothelial cells (1). The partially active single chain can be further processed to full activity by plasmin, tissue kallikrein or Factor Xa. Active PLAT converts plasminogen to plasmin, a fibrinolytic protease, by hydrolyzing an Arg-Val peptide bond in plasminogen. Unusually high levels of tPA activity can result in excessive bleeding, and low levels of tPA activity can result in thrombosis or embolism. Human PLAT contains 4 domains; the N-terminal fibronectin type-1 domain, an epidermal growth factor-like domain, two kringle domains and a serine protease catalytic domain.
- Lijnen H.R. and D. Collen (2004) Handbook of Proteolytic Enzymes (ed. Barrett, et al.) p. 1684, Academic Press, San Diego.
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