Recombinant P. heparinus Chondroitinase AC Protein, CF Summary
Gln23-Lys700 with an N-terminal Met and 6-His tag
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, 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.
- 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
- Dilute rP. heparinus Chondroitinase AC to 1 ng/μL in Assay Buffer.
- Dilute Substrate to 4 mg/mL in Assay Buffer.
- 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.
- Read at an absorbance of 232 nm in kinetic mode for 5 minutes.
- 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:
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).
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