Recombinant Mouse Latexin Protein, CF Summary
Product Specifications
Glu2-Glu222, with an N-terminal Met and 6-His tag
Accession # P70202
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.
3620-PI
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, 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% (v/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Mouse Latexin (rmLatexin) (Catalog # 3620-PI)
- Recombinant Mouse Carboxypeptidase A1/CPA1 (rmCPA1) (Catalog # 2765-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 rmCPA1 to 100 µg/mL with 1.0 µg/mL Trypsin in Assay Buffer.
- Incubate at room temperature for 1 hour.
- Dilute activated rmCPA1 to 20 µg/mL in Assay Buffer.
- Prepare a curve of rmLatexin (MW: 26,297 Da) in Assay Buffer. Make the following serial dilutions: 2500, 1600, 800, 300, 200, 150, 100, 50, 25, and 12.5 nM.
- Mix equal volumes of the rmLatexin curve dilutions and the diluted rmCPA1. Include a control (in duplicate) containing Assay Buffer and the diluted rmCPA1.
- Incubate mixtures at 37 °C for 1 hour.
- After incubation, dilute the mixtures 50 fold in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer containing 200 µM DTNB.
- Load 50 µL of the diluted incubated mixtures to a 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 rmLatexin by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
- The specific activity for rmCPA1 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:
- rmCPA1: 0.010 µg
- rmLatexin curve: 12.5, 8, 4, 1.5, 1.0, 0.75, 0.5, 0.25, 0.125, and 0.0625 nM
- Substrate: 100 μM
- DTNB: 100 μM
Reconstitution Calculator
Background: Latexin
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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