Recombinant Human MAN2B1 Protein, CF Summary
Product Specifications
Gly50-Gly1011, with a C-terminal 6-His tag
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.
9826-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.
|
Assay Procedure
- Assay Buffer: 50 mM Sodium Acetate, pH 4.5
- Recombinant Human MAN2B1 (rhMAN2B1) (Catalog # 9826-GH)
- Substrate: 4-Methylumbelliferyl-alpha -D-mannopyranoside (Sigma, Catalog # M3657), 50 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhMAN2B1 to 2 ng/μL in Assay Buffer.
- Dilute Substrate to 400 µM in Assay Buffer.
- Load 50 μL of 2 ng/μL rhMAN2B1 into the plate, and start the reaction by adding 50 μL of 400 µM Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of 400 µM Substrate.
- Read at excitation and emission wavelengths of 365 nm and 445 nm, respectively, (top read) in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU) |
| amount of enzyme (µg) |
*Adjusted for Substrate Blank.
**Derived using calibration standard 4-Methylumbelliferone (Sigma, Catalog # M1381).
- rhMAN2B1: 0.1 µg
- Substrate: 200 µM
Reconstitution Calculator
Background: MAN2B1
The lysosomal catabolism of N-glycans on mammalian glycoproteins occurs through the sequential exoglycosidase digestion of oligosaccharides from the non‑reducing terminus down to the carbohydrate-peptide core region (1). Included among the hydrolytic activities responsible for this oligosaccharide degradation is a broad specificity exo-alpha -mannosidase MAN2B1 that catalyzes the hydrolysis of alpha 1,2-, alpha 1,3-, and alpha 1,6-mannoside linkages present in complex, hybrid, and high mannose Asn-linked glycans (2, 3, 4). Defects in MAN2B1 result in lysosomal alpha -mannosidosis that shows symptom of mental retardation, recurrent infections, impaired hearing and Hurler-like skeletal changes (5, 6).
Manufacturing Specifications
- Aronson, N.N. Jr. and Kuranda, M.J. (1989) FASEB J. 3:2615.
- Liao, Y.F. et al. (1996) J. Biol. Chem. 271:28348.
- Gotoda, Y. et al. (1989) Am. J. Hum. Genet. 63:1015.
- Nebes, V.L. and Schmidt, M.C. (1994) Biochem. Biophys. Res. Commun. 200:239.
- Nilssen, O, et al. (1997) Hum. Mol. Genet. 6:717.
- Yu, P. et al. (2015) Genet. Med. 17:971.
FAQs
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