Recombinant Human u-Plasminogen Activator/Urokinase, CF

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Recombinant Human u-Plasminogen Activator/Urokinase (Catalog # 1310-SE) is measured by its ability to cleave a peptide substrate,N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC).

Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

Order Details

Recombinant Human u-Plasminogen Activator/Urokinase, CF Summary

Details of Functionality
Measured by its ability to cleave a peptide substrate, N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC). The specific activity is >2,000 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human u-Plasminogen Activator (uPA)/Urokinase protein
Met1-Leu431 with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Ser21, Ile179 & Lys156
Protein/Peptide Type
Recombinant Enzymes
Gene
PLAU
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
18 kDa (A chain), 30 kDa (B chain).
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
18 kDa and 32 kDa, reducing conditions
Publications
Read Publications using
1310-SE in the following applications:

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in HEPES, NaCl and CaCl2.
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Assay Procedure
  • Assay Buffer: 50 mM Tris, 0.01% (v/v) Tween® 20, pH 8.5
  • Recombinant Human u‑Plasminogen Activator (uPA)/Urokinase (rhuPA)  (Catalog # 1310-SE)
  • 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
  1. Dilute rhuPA to 1 ng/µL in Assay Buffer.
  2. Dilute Substrate to 200 µM in Assay Buffer.
  3. Load 50 µL of the 1 ng/µL rhuPA 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 rhuPA.
  4. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. 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 (Sigma, Catalog # A-9891)

Per Well:
  • rhuPA: 0.05 µg
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human u-Plasminogen Activator/Urokinase, CF

  • ATF
  • EC 3.4.21
  • EC 3.4.21.73
  • plasminogen activator, urokinase
  • PLAU
  • uPA
  • u-PA
  • uPlasminogen Activator
  • u-Plasminogen Activator
  • urinary
  • Urokinase
  • urokinase-type plasminogen activator

Background

uPA is a serine protease with an extremely limited substrate specificity, cleaving the sequence Cys-Pro-Gly-Arg560-Val561-Val-Gly-Gly-Cys in plasminogen to form plasmin (1). uPA is a potent marker of invasion and metastasis in a variety of human cancers associated with breast, stomach, colon, bladder, ovary, brain and endometrium (2). For example, the combination (both low vs. either or both high) of uPA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), outperforms the single factors as well as other traditional prognostic factors with regard to risk group assessment for breast cancer, particularly in node-negative breast cancer (3). The human uPA is initially synthesized as 431 amino acid precursor with a N-terminal signal peptide (20 residues) (4‑6). The single chain molecule is processed into a disulfide-linked two-chain molecule. The B chain starting at Ile179 corresponds to the catalytic domain. Two forms of the A chain exist, one starting at Ser21 (the long form) and the other at Lys156 (the short form). The resulting two-chain forms have different molecular weights (MW). The B chain is common for both forms whereas the long and short A chains are unique to the high and low MW forms, respectively. The long A chain contains an EGF-like domain, which is responsible for binding of the uPA receptor (uPAR). Both high and low MW forms exist in the purified recombinant human uPA.

  1. Ellis, V. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. eds., Academic Press, San Diego, pp.1677.
  2. Duffy, M.J. (2002) Biochem. Soc. Trans. 30:207.
  3. Harbeck, N. et al. (2002) Clin. Breast Cancer 3:196.
  4. Riccio, A. et al. (1985) Nucleic Acids Res. 13:2785.
  5. Nagai, M. et al. (1985) Gene 36:183.
  6. Jacobs, P. et al. (1985) DNA 4:139.

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1310-SE
Species: Hu
Applications: Enzyme Activity

Publications for u-Plasminogen Activator/Urokinase (1310-SE)(7)

We have publications tested in 3 confirmed species: Human, Mouse, N/A.

We have publications tested in 3 applications: Bioassay, ELISA Standard, Enzyme Assay.


Filter By Application
Bioassay
(5)
ELISA Standard
(1)
Enzyme Assay
(1)
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Filter By Species
Human
(3)
Mouse
(2)
N/A
(1)
All Species
Showing Publications 1 - 7 of 7.
Publications using 1310-SE Applications Species
Pant S, Mukonoweshuro A, Desai B, Ramjee M, Selway C, Tarver G, Wright A, Birchall K, Chapman T, Tervonen T, Klefstrom J Design, Synthesis, and Testing of Potent, Selective Hepsin Inhibitors via Application of an Automated Closed-Loop Optimization Platform. J Med Chem, 2018;61(10):4335-4347. 2018 [PMID: 29701962] (Enzyme Assay) Enzyme Assay
X Han, PD Bryson, Y Zhao, GE Cinay, S Li, Y Guo, N Siriwon, P Wang Masked Chimeric Antigen Receptor for Tumor-Specific Activation Mol. Ther, 2017;25(1):274-284. 2017 [PMID: 28129121] (Bioassay, Human) Bioassay Human
Senescent peritoneal mesothelium creates a niche for ovarian cancer metastases Cell Death Dis, 2016;7(12):e2565. 2016 [PMID: 28032864] (Bioassay, Human) Bioassay Human
Doni A, Musso T, Morone D, Bastone A, Zambelli V, Sironi M, Castagnoli C, Cambieri I, Stravalaci M, Pasqualini F, Laface I, Valentino S, Tartari S, Ponzetta A, Maina V, Barbieri S, Tremoli E, Catapano A, Norata G, Bottazzi B, Garlanda C, Mantovani A An acidic microenvironment sets the humoral pattern recognition molecule PTX3 in a tissue repair mode. J Exp Med, 2015;212(6):905-25. 2015 [PMID: 25964372] (Bioassay, Mouse) Bioassay Mouse
Lebeau A, Sevillano N, Markham K, Winter M, Murphy S, Hostetter D, West J, Lowman H, Craik C, VanBrocklin H Imaging active urokinase plasminogen activator in prostate cancer. Cancer Res, 2015;75(7):1225-35. 2015 [PMID: 25672980] (Bioassay, Human) Bioassay Human
Zhuo, Jingli, Tan, Ee Hong, Yan, Benedict, Tochhawng, Lalchhan, Jayapal, Manikand, Koh, Shiuan, Tay, Hwee Kee, Maciver, Sutherla, Hooi, Shing Ch, Salto-Tellez, Manuel, Kumar, Alan Pre, Goh, Yaw Chon, Lim, Yaw Chyn, Yap, Celestia Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade. PLoS ONE, 2012;7(8):e43594. 2012 [PMID: 22927998] (ELISA Standard, N/A) ELISA Standard N/A
Endo-Munoz L, Cumming A, Rickwood D, Wilson D, Cueva C, Ng C, Strutton G, Cassady AI, Evdokiou A, Sommerville S, Dickinson I, Guminski A, Saunders NA Loss of osteoclasts contributes to development of osteosarcoma pulmonary metastases. Cancer Res., 2010;70(18):7063-72. 2010 [PMID: 20823153] (Bioassay, Mouse) Bioassay Mouse

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Bioinformatics

Gene Symbol PLAU
Uniprot