Recombinant Human Latexin Protein, CF Summary
Glu2-Glu222, with an N-terminal Met and 6-His tag
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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 and NaCl.|
|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 Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.05% Brij-35, pH 7.5 (TCNB)
- Recombinant Human Latexin (rhLatexin) (Catalog # 3246-PI)
- Recombinant Human Carboxypeptidase A1/CPA1 (rhCPA1) (Catalog # 2856-ZN)
- Trypsin (Sigma, Catalog # T-1426)
- Substrate: Ac-Phe-Thiaphe-OH (Peptides International, Catalog # STP-3621-PI),10 mM stock in DMSO
- 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB), (Sigma, Catalog # D-8130) 10 mM stock in DMSO
- 96 well clear plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhCPA1 to 100 µg/mL with 1 µg/mL Trypsin in Assay Buffer.
- Incubate at room temperature for 1 hour.
- Dilute active rhCPA1 to 20 µg/mL in Assay Buffer.
- Prepare a curve of rhLatexin (MW: 26,575 Da) in assay buffer. Make the following serial dilutions: 1600, 800, 400, 200, 150, 100, 50, and 25 nM.
- Mix equal volumes of the rhLatexin curve dilutions and the diluted rhCPA-1. Include a control (in duplicate) containing Assay Buffer and the diluted rhCPA-1.
- Incubate mixtures at 37 °C for 1 hour.
- After incubation, dilute the mixtures by 50 fold in Assay Buffer.
- Combine equal volumes of Substrate and DTNB. Dilute this mixture to 200 µM Substrate and 200 µM DTNB in Assay Buffer.
- Load 50 µL of the incubated mixtures into the plate, and start the reaction by adding 50 µL of 200 µM Substrate mixture.
- Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
- Derive the 50% inhibition concentration (IC50) for rhLatexin by plotting OD/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for rhCPA1 at each point may be determined using the following formula (if needed):
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol|
|ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Using the extinction coefficient 13260 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD Per Well:
- rhCPA1: 0.010 µg
- rhLatexin curve: 8, 4, 2.0, 1.0, 0.75, 0.5, 0.25, and 0.125 nM.
- Substrate: 100 µM
- DTNB: 100 µM
Human Latexin, also known as tissue carboxypeptidase inhibitor, is the only mammalian carboxypeptidase inhibitor identified so far. Latexin was initially identified as a marker of neurons in the lateral neocortex of the developing brain (hence latexin) (1). Further studies have shown that latexin is highly expressed in mast cells and macrophages (2, 3). It is induced in acute pancreatitis and lung inflammatory disease. In addition, it is able to inhibit carboxypeptidases from the pancreas (CPA1 and CPA2) and mast cells (CPA3) (4). Therefore, it is thought that latexin primarily functions in inflammation and innate immunity pathways. Compared to other carboxypeptidase inhibitors from plants and parasites, the 222 amino acid latexin is much larger and more importantly, it lacks some conserved C-terminal residues, which interact with the target carboxypeptidase in a substrate-like manner (5). This distinct feature of latexin suggests that it has a different carboxypeptidase inhibition mechanism.
- Arimatsu, Y. et al. (1994) Neurosci. Res. 20:131.
- Uratani, Y. et al. (2000) Biochem. J. 346:817.
- Aagaard, A. et al. (2005) Structure 13:309.
- Normant, E. et al. (1995) Proc. Natl. Acad. Sci. USA 92:12225.
- Reverter, D. et al. (2000) Nat. Struct. Biol. 7:322.
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