Recombinant Mouse Kell Protein, CF Summary
Phe50-Trp713, with an N-terminal 7-His tag
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 Tris, NaCl and ZnCl2.|
|Shipping||The product is shipped with dry ice or equivalent. 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: 0.1 M MES, pH 6.5
- Recombinant Mouse Kell (rmKell) (Catalog # 1454-ZN)
- Substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # ES005), 2 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmKell to 20 ng/µL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the 20 ng/µL rmKell in a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 1 hour.
- 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
- rmKell: 1 µg
- Substrate: 10 µM
Kell, a type II membrane glycoprotein, is linked through a single disulfide bond to XK, a putative membrane transporter. The two proteins constitute the Kell blood group antigens (1). Kell is a zinc endopeptidase of the neprilysin (NEP) family which also includes endothelin converting enzymes (ECE-1 and ECE-2), PEX, XCE, DINE and several NEP-like proteins (2). It has been shown to cleave big endothelin-3 (ET-3) at Trp21-Ile22, yielding ET-3, and to a much lesser extent, big ET-1 and big ET-2 at Trp21-Val22, yielding ET-1 and ET-2 (3). Several different molecular defects cause the Kell null phenotype, which has no obvious clinical outcome (4).
- Lee, S. et al. (2000) Transfus. Med. Rev. 14:93.
- Turner, A.J. et al. (2001) BioEssays. 23:261.
- Lee, S. et al. (1999) Blood 94:1440.
- Lee, S. et al. (2001) J. Biol. Chem. 276:27281.
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Fluorogenic Peptide Substrates
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