Human alpha 2-Macroglobulin Protein, CF Summary
Product Specifications
The human plasma used for the isolation of this product was certified by the supplier to be HIV-1 and HBsAg negative at the time of shipment. Human blood products should always be treated in accordance with universal handling precautions.
Analysis
Product Datasheets
Carrier Free
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.
1938-PI
| Formulation | Supplied as a 0.2 μm filtered solution in NaH2PO4, NaCl and Glycerol. |
| 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 Procedure
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Human alpha 2-Macroglobulin (h alpha 2-Macroglobulin) (Catalog # 1938-PI)
- Recombinant Human Serpin F2 (rhSerpin F2) (Catalog # 1470-PI)
- Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE)
- Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhTrypsin 3 to 1.28 µg/mL with Assay Buffer.
- Prepare a curve of h alpha 2-Macroglobulin with Assay Buffer. Dilute h alpha 2-Macroglobulin (MW: 720000 Da) to the following concentrations: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 0.3125 nM.
- Combine 30 µL of h alpha 2-Macroglobulin curve with 30 µL of 1.28 µg/mL rhTrypsin 3. Include a control containing 30 µL of Assay Buffer with 30 µL of rhTrypsin 3 in duplicate. Also include a h alpha 2-Macroglobulin control in duplicate for each sample tested containing 30 µL of 200 nM h alpha 2-Macroglobulin and 30 µL Assay Buffer.
- Incubate at 37 °C for 1 hour.
- Dilute rhSerpin F2 to 40 µg/mL with Assay Buffer.
- Add 60 µL of 40 µg/mL rhSerpin F2 to each reaction.
- Incubate at 37 °C for 15 minutes.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load into plate 50 µL of h alpha 2-Macroglobulin curve containing rhTrypsin 3 and rhSerpin F2, and start the reaction by adding 50 µL of 20 µM Substrate to each well.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration (IC50) of h alpha 2-Macroglobulin, by its trapping of rhTrypsin 3, toward rhSerpin F2 activity by plotting RFU/min (or specific activity) vs. concentration (h alpha 2-Macroglobulin) with 4-PL fitting.
- The specific activity for rhTrypsin 3 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 h alpha 2-Macroglobulin control
**Derived using calibration MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
Per Well:- h alpha 2-Macroglobulin: 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, and 0.0391 nM
- rhTrypsin 3: 0.016 µg
- rhSerpin F2: 1 µg
- Substrate: 10 µM
Reconstitution Calculator
Background: alpha 2-Macroglobulin
Human alpha 2-macroglobulin (h alpha 2M) is a serum glycoprotein that has sequence similarity to other members of the alpha 2M family including complement components C3, C4 and C5 (1). alpha 2M is synthesized as a polypeptide of 1474 amino acids with a signal peptide (23 residues) (2). The mature protein is a tetramer (720 kDa) of 4 identical subunits (180 kDa), which form two disulfide bond-linked dimers. As a general and irreversible protease inhibitor implicated in many processes, alpha 2M is able to inhibit all four classes of proteases by a unique trapping mechanism. The bait region of h alpha 2M (residues 690‑728) contains specific cleavage sites for different proteases. The cleavage of the bait region by a protease induces a conformation change in alpha 2M, which then traps and forms a covalent bond with the protease. The trapped protease remains active against small peptide substrates but loses its ability to interact with large protein substrates or inhibitors.
- Sottrup-Jensen, L. et al. (1985) Proc. Natl. Acad. Sci. USA 82:9.
- Kan, C.C. et al. (1985) Proc. Natl. Acad. Sci. USA 82:2282.
Citations for Human alpha 2-Macroglobulin 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.
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Citations: Showing 1 - 5
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Post-Translational Regulation And Proteolytic Activity Of The Metalloproteinase Adamts8
Authors: S Santamaria, DR Martin, X Dong, K Yamamoto, SS Apte, J Ahnström
The Journal of Biological Chemistry, 2021-10-21;0(0):101323.
Species: Human
Sample Types:
Applications: Bioassay -
Exosomes from acellular Wharton's jelly of the human umbilical cord promotes skin wound healing
Authors: N Bakhtyar, MG Jeschke, E Herer, M Sheikholes, S Amini-Nik
Stem Cell Res Ther, 2018-07-13;9(1):193.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Effects of sFlt-1 and alpha 2-macroglobulin on vascular endothelial growth factor-induced endothelin-1 upregulation in human microvascular endothelial cells.
Authors: Yi K, Jung S, Cho G, Seol H, Hong S, Oh M, Kim H
Placenta, 2013-09-25;35(1):64-9.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Regulated proteolytic processing of Reelin through interplay of tissue plasminogen activator (tPA), ADAMTS-4, ADAMTS-5, and their modulators.
Authors: Krstic D, Rodriguez M, Knuesel I
PLoS ONE, 2012-10-17;7(10):e47793.
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay -
Enzymatic properties of human kallikrein-related peptidase 12 (KLK12).
Authors: Memari</LastName><ForeNam N</Initial, Memari N, Jiang W, Diamandis EP, Luo LY
Biol. Chem., 2007-04-01;388(4):427-35.
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay
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