Recombinant Human Serpin E1/PAI-1 Protein, CF

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Recombinant Human Serpin E1/PAI-1 (Catalog # 1786-PI) is measuredby its ability to inhibit uPA cleavage of a peptide substrate,N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC).

Product Details

Summary
Reactivity HuSpecies Glossary
Applications Inhibition Activity
Format
Carrier-Free

Order Details

Recombinant Human Serpin E1/PAI-1 Protein, CF Summary

Details of Functionality
Measured by its ability to inhibit uPA cleavage of a peptide substrate, N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC). The IC50 value is <13 nM, as measured under the described conditions.
Source
Spodoptera frugiperda, Sf 21 (stably transfected)-derived human Serpin E1/PAI-1 protein
Met1-Pro402, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Ser22 & Val24
Protein/Peptide Type
Recombinant Enzymes
Gene
SERPINE1
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
44 kDa.
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
43 kDa, reducing conditions
Publications
Read Publications using
1786-PI 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 reconstitution.
Buffer
Lyophilized from a 0.2 μm filtered solution in Sodium Acetate, NaCl and CHAPS.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Reconstitution Instructions
Reconstitute at 500 μg/mL in sterile 50 mM Sodium Acetate and 100 mM NaCl, pH 5.5.
Assay Procedure
  • Assay Buffer: 50 mM Tris, 0.01% (v/v) Tween® 20, pH 8.5
  • Recombinant Human Serpin E1/PAI-1 (rhSerpin E1/PAI-1) (Catalog # 1786-PI)
  • Recombinant Human u‑Plasminogen Activator (uPA)/Urokinase (rhuPA) (Catalog # 1310-SE)
  • Substrate: Z-Gly-Gly-Arg-AMC (Bachem, Catalog # I-1140)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhuPA to 2 µg/mL in Assay Buffer.
  2. Prepare a rhSerpin E1/PAI-1 (MW: 44146 Da) dilution curve in Assay Buffer. Make the following serial dilutions:  1500, 750, 375, 187.5, 125, 75, 50, 25, and 12.5 nM.
  3. Mix equal volumes of the rhSerpin E1/PAI-1 curve and the diluted rhuPA at 2 µg/mL. Include 2 controls containing rhuPA and Assay Buffer only.
  4. Incubate mixtures at room temperature for 15 minutes.
  5. After incubation, dilute the mixtures 5 fold in Assay Buffer.
  6. Dilute the Substrate to 200 µM in Assay Buffer.
  7. Load 50 µL of the incubated reactions into a plate, and start the reaction by adding 50 µL of 200 µM Substrate to each well.
  8. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  9. Determine the 50% inhibiting concentration (IC50) for rhSerpin E1 by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
  10. The specific activity for rhuPA at each point may be determined using the following formula (if necessary):

     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 # A-9891).

Per Well:
  • rhuPA: 0.010 µg
  • rhSerpin E1/PAI-1 curve: 75, 37.5, 18.75, 9.375, 6.25, 3.75, 2.5, 1.25, and 0.625 nM
  • 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 Serpin E1/PAI-1 Protein, CF

  • Endothelial plasminogen activator inhibitor
  • Nexin
  • PAI1
  • PAI-1
  • PAI1PAI-1
  • PAISerpin E1
  • PLANH1
  • PLANH1plasminogen activator inhibitor 1
  • serine (or cysteine) proteinase inhibitor, clade E (nexin, plasminogenactivator inhibitor type 1), member 1
  • Serpin E1
  • serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitortype 1), member 1

Background

As a member of the Serpin superfamily of serine protease inhibitors, Serpin E1/PAI‑1 is the principal inhibitor of urokinase‑type plasminogen activator (uPA) and tissue‑type PA (1, 2). As important regulators of extracellular matrix remodeling, uPA and PAI‑1 play a major role in many processes such as angiogenesis, tumor invasion and obesity (2‑4). For example, uPA and PAI-1 are the only tumor prognostic factors validated at the highest level of evidence with regard to their clinical utility in breast cancer (5). The human PAI-1 is initially synthesized as 402 amino acid precursor with a N‑terminal signal peptide (6, 7). PAI‑1 may exist in one of two possible conformations, designated as active or latent (8). The purified rhPAI‑1 is active against rhuPA. The heterogeneity at the N‑terminus of the purified
rhPAI‑1 has been observed before for both the recombinant and native proteins (9).

  1. Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
  2. Stefansson, S. et al. (2003) Curr. Pharm. Des. 9:1545.
  3. Duffy, M.J. (2002) Clin. Chem. 48:1194.
  4. Juhan-Vague, I. et al. (2003) J. Thromb. Haemost. 1:1575.
  5. Harbeck, N. et al. (2002) Clin. Breast Cancer 3:196.
  6. Pannekoek, H. et al. (1986) EMBO J. 5:2539.
  7. Ginsburg, D. et al. (1986) J. Clin. Invest. 78:1673.
  8. Wang, Z. et al. (1996) Biochemistry 35:16443.
  9. Stromqvist, M. et al. (1994) Protein Expr. Purif. 5:309.

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Publications for Serpin E1/PAI-1 (1786-PI)(9)

We have publications tested in 2 confirmed species: Human, N/A.

We have publications tested in 2 applications: Bioassay, Enzyme Assay.


Filter By Application
Bioassay
(6)
Enzyme Assay
(2)
All Applications
Filter By Species
Human
(7)
N/A
(1)
All Species
Showing Publications 1 - 9 of 9.
Publications using 1786-PI Applications Species
A Infante, CI Rodríguez Secretome analysis of in vitro aged human mesenchymal stem cells reveals IGFBP7 as a putative factor for promoting osteogenesis Sci Rep, 2018;8(1):4632. 2018 [PMID: 29545581] (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, N/A) Bioassay N/A
Santhekadur P, Akiel M, Emdad L, Gredler R, Srivastava J, Rajasekaran D, Robertson C, Mukhopadhyay N, Fisher P, Sarkar D Staphylococcal nuclease domain containing-1 (SND1) promotes migration and invasion via angiotensin II type 1 receptor (AT1R) and TGFbeta signaling. FEBS Open Bio, 2014;4(0):353-61. 2014 [PMID: 24918049] (Bioassay, Human) Bioassay Human
Krstic D, Rodriguez M, Knuesel I Regulated proteolytic processing of Reelin through interplay of tissue plasminogen activator (tPA), ADAMTS-4, ADAMTS-5, and their modulators. PLoS ONE, 2012;7(10):e47793. 2012 [PMID: 23082219] (Enzyme Assay, Human) Enzyme Assay Human
Schipper HS, De Jager W, van Dijk ME, Meerding J, Zelissen PM, Adan RA, Prakken BJ, Kalkhoven E A multiplex immunoassay for human adipokine profiling. Clin. Chem., 2010;56(0):1320. 2010 [PMID: 20530731]
Huang MC, Patel K, Taub DD, Longo DL, Goetzl EJ Human CD4- 8- T cells are a distinctive immunoregulatory subset. FASEB J, 0;24(7):2558-66. 0 [PMID: 20154266] (Bioassay, Human) Bioassay Human
Szabo R, Hobson JP, List K, Molinolo A, Lin CY, Bugge TH Potent Inhibition and Global Co-localization Implicate the Transmembrane Kunitz-type Serine Protease Inhibitor Hepatocyte Growth Factor Activator Inhibitor-2 in the Regulation of Epithelial Matriptase Activity. J. Biol. Chem., 2008;283(43):29495-504. 2008 [PMID: 18713750] (Bioassay, Human) Bioassay Human
Memari&lt;/LastName&gt;&lt;ForeNam N&lt;/Initial, Memari N, Jiang W, Diamandis EP, Luo LY Enzymatic properties of human kallikrein-related peptidase 12 (KLK12). Biol. Chem., 2007;388(4):427-35. 2007 [PMID: 17391064] (Enzyme Assay, Human) Enzyme Assay Human

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Bioinformatics

Gene Symbol SERPINE1
Uniprot