Recombinant Human Latexin Protein, CF

Catalog #: 3246-PI Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
3246-PI-010

R&D Systems Recombinant Proteins and Enzymes

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Recombinant Human Latexin 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 carboxypeptidase A1 cleavage of the colorimetric peptide substrate Ac-Phe-Thiaphe-OH in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. The IC₅₀ value is <1 nM, as measured under the described conditions.

Source

E. coli-derived human Latexin protein
Glu2-Glu222, with an N-terminal Met and 6-His tag

Accession #

AAH05346

N-terminal Sequence
Analysis

Met

Predicted Molecular Mass

27 kDa

SDS-PAGE

30 kDa, reducing conditions

Product Datasheets

3246-PI

Product Datasheet

COA

SDS

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.

3246-PI

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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

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 V_max* (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

Reconstitution Calculator

Background: Latexin

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.

References