Recombinant Human Serpin A1/alpha-1-Antitrypsin Protein, CF

Catalog # Availability Size / Price Qty
1268-PI-010
Recombinant Human Serpin A1/alpha-1-Antitrypsin Protein Enzyme Activity
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Product Details
Citations (3)
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Recombinant Human Serpin A1/alpha-1-Antitrypsin Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002). The IC50 value is approximately <5.0 nM, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Serpin A1/alpha 1-Antitrypsin protein
Glu25-Lys418, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Analysis
Glu25
Predicted Molecular Mass
46 kDa
SDS-PAGE
60 kDa, reducing conditions

Product Datasheets

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1268-PI

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

1268-PI

Formulation Lyophilized from a 0.2 μm filtered solution in Tris, NaCl and CaCl2 with Trehalose.
Reconstitution Reconstitute at 100 μg/mL in sterile 50 mM Tris, 10 mM CaCl2 and 150 mM NaCl, pH 7.5.
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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.

Assay Procedure

Materials
  • Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Human Serpin A1/ alpha 1‑Antitrypsin (rhSerpin A1) (Catalog # 1268-PI)
  • Trypsin (Sigma, Catalog # T1426)
  • Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002), 2 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 Trypsin to 0.25 µg/mL in Assay Buffer.
  2. Prepare a curve of rhSerpin A1 (MW: 45,667 Da) in Assay Buffer.  Make the following serial dilutions:
    200 nM, 100 nM, 50 nM, 33.3 nM, 22.2 nM, 14.8 nM, 9.88 nM, and 3.29 nM.
  3. Combine 25 µL of 0.25 µg/mL Trypsin with 25 µL of rhSerpin A1 serial curve dilutions. Include two controls of 25 µL Assay Buffer with 25 µL of 0.25 µg/mL Trypsin.
  4. Incubate at room temperature for 30 minutes.
  5. After incubation, add 200 µL of Assay Buffer to each serial curve dilution.
  6. Dilute Substrate to 20 µM in Assay Buffer.
  7. In a plate, load 50 µL of the diluted rhSerpin A1 curve, and start the reaction by adding 50 µL of 20 µM Substrate to wells.
  8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  9. Derive the 50% inhibition concentration (IC50) value for hSerpin A1 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  10. The specific activity for Trypsin at each point may be determined using the following formula (if needed):

     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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:
  • Trypsin: 0.00125 µg
  • rhSerpin A1: 10, 5, 2.5, 1.665, 1.11, 0.74, 0.494, 0.165 and 0 nM
  • Substrate: 10 µM

Scientific Data

Enzyme Activity Recombinant Human Serpin A1/alpha-1-Antitrypsin Protein Enzyme Activity View Larger

Recombinant Human Serpin A1/alpha-1-Antitrypsin (Catalog # 1268-PI) is measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002).

Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Serpin A1/alpha 1-Antitrypsin

Serpin A1 is the archetypal member of the Serpin superfamily of the serine protease inhibitors (1). As one of the most abundant proteinase inhibitors in the circulation, it is synthesized in the liver and secreted into the bloodstream with the major function to protect tissues against neutrophil elastase. A severe serpin A1 deficiency leads to several clinical complications such as pulmonary emphysema, juvenile hepatitis, cirrhosis, and hepatocellular carcinoma (2). The deficiency is caused by point mutations in naturally occurring serpin A1 variants (over 70 are known). For example, the Z variant (Glu342 to Lys) forms intracellular inclusion bodies, is not secreted, and leads to a severe serpin A1 deficiency (3).

References
  1. Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
  2. Barbour, K.W. et al. (2002) Genomics 80:515.
  3. Lomas, D.A. et al. (2002) Biochem. Soc. Trans. 30:89.
Entrez Gene IDs
5265 (Human)
Alternate Names
A1A; A1AT; AATMGC23330; alpha 1-Antitrypsin; alpha 1-Proteinase Inhibitor; Alpha-1 protease inhibitor; Alpha-1-antiproteinase; alpha-1-antitrypsin; antitrypsin), member 1; member 1; PIMGC9222; serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase; serine (or cysteine) proteinase inhibitor, clade A, member 1; Serpin A1; serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin)

Citations for Recombinant Human Serpin A1/alpha-1-Antitrypsin Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

3 Citations: Showing 1 - 3
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  1. Mutations in lectin complement pathway genes COLEC11 and MASP1 cause 3MC syndrome.
    Authors: Rooryck C, Diaz-Font A, Osborn DP
    Nat. Genet., 2011;43(3):197-203.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  2. Enzymatic properties of human kallikrein-related peptidase 12 (KLK12).
    Authors: Memari&lt;/LastName&gt;&lt;ForeNam N&lt;/Initial, Memari N, Jiang W, Diamandis EP, Luo LY
    Biol. Chem., 2007;388(4):427-35.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Enzyme Assay
  3. Mannan-binding lectin-associated serine protease 3 cleaves synthetic peptides and insulin-like growth factor-binding protein 5.
    Authors: Cortesio CL, Jiang W
    Arch. Biochem. Biophys., 2006;449(1):164-70.
    Species: N/A
    Sample Types: Protein
    Applications: Enzyme Assay

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Fluorogenic Peptide Substrates

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Recombinant Human Serpin A1/alpha-1-Antitrypsin Protein, CF
By Balaji Mahender on 12/20/2017
Application: Immunoassay Standard