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Recombinant P. heparinus Chondroitinase AC Protein, CF

Catalog #: 8384-GH Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
8384-GH-050
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Recombinant P. heparinus Chondroitinase AC Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Endotoxin Level
<0.01 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to hydrolyze chondroitin sulfate. The specific activity is >50,000 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived p. heparinus Chondroitinase AC protein
Gln23-Lys700 with an N-terminal Met and 6-His tag
Accession #
Q59288
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
78 kDa
SDS-PAGE
66 kDa, reducing conditions

Product Datasheets

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8384-GH

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.

8384-GH

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35.
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: 25 mM Tris, 50 mM NaCl, 10 mM MgCl2, pH 7.5
  • Recombinant P. heparinus Chondroitinase AC (rP. heparinus Chondroitinase AC) (Catalog # 8384-GH)
  • Substrate: Chondroitin Sulfate (Sigma, Catalog # C6737), 50 mg/mL stock in deionized water
  • 96 well UV Plate (Costar, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rP. heparinus Chondroitinase AC to 1 ng/μL in Assay Buffer.
  2. Dilute Substrate to 4 mg/mL in Assay Buffer.
  3. Load 50 μL of 1 ng/μL rP. heparinus Chondroitinase AC into the plate, and start the reaction by adding 50 μL of 4 mg/mL Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of 4 mg/mL Substrate.
  4. Read at an absorbance of 232 nm in kinetic mode for 5 minutes.
  5. Calculate specific activity:

     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 3800 M-1cm-1
   ***Using the path correction 0.32 cm
   Note: the output of many spectrophotometers is in mOD. Per Well:
  • rP. heparinus Chondroitinase AC: 0.050 μg
  • Chondroitin Sulfate: 200 μg
      • 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: Chondroitinase AC

      Chondroitinase AC from P. heparinum is a depolymerizing lyase that degrades chondroitin sulfates A and C, but not chondroitin sulfate B (1). Both chondroitin sulfate A and C contain D-glucuronic acid, N-acetylgalactosamine, and sulfate residues in equal molar quantities (2). The sulfate ester is at the 4-O position of N-acetylgalactosamine residues on chondroitin sulfate A and at the 6-O position of N-acetylgalactosamine residues on chondroitin sulfate C. In contrast chondroitin sulfate B contains L-iduronic acid instead of glucuronic acid (3). Chondroitinase AC cleaves, via an elimination mechanism, chondroitin sulfate chains between N-acetylgalactosamine and glucuronic acid residues. The reaction yields oligosaccharide products, primarily disaccharides, containing unsaturated uronic acids that can be detected by UV spectroscopy. The enzyme shows approximately equal activity with chondroitin sulfates A orC (3). Chondrotinase AC can be used to specifically degrade chondroitin A and C and distinguish among different species of glycosaminoglycans (4).

      References
      1. Tkalec, A.L. et al. (2000) Appl. Environ. Microbiol. 66:29.
      2. Saito, H., et al. (1968) J. Biol. Chem. 243:1536.
      3. Gu, K. et al. (1995) Biochem. J. 312:569.
      4. Wu, Z.L. et al. (2011) Glycobiology 21:625.
      Entrez Gene IDs
      8251875 (P. heparinus)
      Alternate Names
      Chondroitinase AC

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