Recombinant Mouse Chitotriosidase/CHIT1 Protein, CF

Catalog # Availability Size / Price Qty
5325-GH-010
R&D Systems Recombinant Proteins and Enzymes
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Recombinant Mouse Chitotriosidase/CHIT1 Protein, CF Summary

Product Specifications

Purity
>90%, 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 cleave 4-Methylumbelliferyl-beta -D-N,N’,N”-triacetylchitotriose (4-MU-TACT). The specific activity is >400 pmol/min/μg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived mouse Chitotriosidase/CHIT1 protein
Ala22-Ser464 (major) & Lys23-Ser464 (minor), both with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Analysis
Ala22 & Lys23
Predicted Molecular Mass
50 kDa
SDS-PAGE
54-57 kDa doublet, reducing conditions

Product Datasheets

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.

5325-GH

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: 10 mM MES, 0.05% (w/v) Brij-35, pH 6.0
  • Reading Buffer: 100 mM Tris, pH 9.0
  • Recombinant Mouse Chitotriosidase/CHIT1 (rmCHIT1) (Catalog # 5325-GH)
  • Substrate: 4-Methylumbelliferyl beta -D-N’,N”-triacetylchitotrioside (4-MU-TACT) (Sigma, Catalog # M-5639), 5 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmCHIT1 to 2 µg/mL in Assay Buffer.
  2. Dilute Substrate to 200 µM in Assay Buffer.
  3. Mix 20 µL of 2 µg/mL of rmCHIT1 and 20 µL of 200 µM of Substrate. Include a Substrate Blank containing 20 µL Assay Buffer and 20 µL Substrate.
  4. Incubate at room temperature for 10 minutes. Protect from light.
  5. Load 90 µL of Reading Buffer per sample into a plate.
  6. Add 10 µL from each reaction mixture into wells containing Reading Buffer.
  7. Read at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in endpoint mode.
  8. Calculate specific activity:

     Specific Activity (pmoles/min/µg) =

Adjusted Fluorescence* (RFU) x Conversion Factor** (pmole/RFU)
Incubation time (min) x amount of enzyme (µg)

     *Adjusted for Substrate Blank
     **Derived using calibration standard 4-Methylumbelliferone (4-MU) (Sigma, Catalog # M1381).

Per Well:
  • rmCHIT1: 0.01 µg
  • Substrate: 10 µM
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Chitotriosidase/CHIT1

Chitotriosidase, encoded by the mouse CHIT1 gene, is a typical member of the chitinase family (1). It is distinct from another member known as acidic mammalian chitinase (AMC), encoded by the CHIA gene, in several aspects. AMC/CHIA is expressed mainly in alveolar macrophages and in both the mouse and human gastrointestinal tract (2). CHIT1, however, is expressed exclusively by phagocytes in humans and in the gastrointestinal tract, tongue, fore-stomach, and Paneth cells of the small intestine in mice (2). Both CHIA and CHIT1 are secreted as 50 kDa proteins. In contrast to CHIA, CHIT1 is not stable under acidic pH and can be processed into a C-terminally truncated 39 kDa form (2‑4). Human CHIT1 is the best of three biomarkers in the monitoring of Gaucher disease (5). The other two commonly used markers include acid phosphatase and angiotensin-converting enzyme (ACE) (5). Human CHIT1 is also a specific marker of macrophage activation in acute ischemic stroke (6).

References
  1. Zheng, T. et al. (2005) Gene 357:37.
  2. Boot, R.G. et al. (2006) J. Histochem. Cytochem. 53:1283.
  3. Boot, R.G. et al. (2001) J. Biol. Chem. 276:6770.
  4. Renkema, G.H. et al. (1997) Eur. J. Biochem. 244:279.
  5. Vellodi, A. et al. (2005) J. Inherit. Metab. Dis. 28:585.
  6. Sotgiu, S. et al. (2005) Eur. Neurol. 54:149.
Entrez Gene IDs
1118 (Human)
Alternate Names
CHI3; CHIT1; CHITCHI3; chitinase 1 (chitotriosidase); chitinase-1; Chitotriosidase; chitotriosidase-1; EC 3.2.1.14; FLJ00314; MGC125322; plasma methylumbelliferyl tetra-N-acetylchitotetraoside hydrolase

Citation for Recombinant Mouse Chitotriosidase/CHIT1 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.

1 Citation: Showing 1 - 1

  1. Spontaneous Chitin Accumulation in Airways and Age-Related Fibrotic Lung Disease
    Authors: SJ Van Dyken, HE Liang, RP Naikawadi, PG Woodruff, PJ Wolters, DJ Erle, RM Locksley
    Cell, 2017;169(3):497-509.e13.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo

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