Recombinant X. campestris beta(1-3)-Galactosidase, CF

• Reactivity: Xc
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant X. campestris beta(1-3)-Galactosidase, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic substrate, 4-Methylumbelliferyl-beta -D-galactopyranoside. The specific activity is >14,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived x. campestris beta (1-3)-Galactosidase protein
  Ser25-Glu613 with an N-terminal Met and 6-His tag
  Accession # NP_638243
• Accession #: NP_638243
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 67 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 63 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 0.1 M MES, pH 5.5
  • Recombinant X. campestris beta (1‑3)-Galactosidase (rXc beta -Galactosidase) (Catalog # 5704-GH)
  • Substrate: 4-methylumbelliferyl-beta -D-galactopyranoside (Sigma, Catalog # M1633), 10 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 rXc beta -Galactosidase to 1 ng/µL in Assay Buffer.
  2. Dilute Substrate to 400 µM in Assay Buffer.
  3. Load into plate 50 µL of 1 ng/µL rXc beta -Galactosidase, and start the reaction by adding 50 µL of 400 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL 400 µM Substrate.
  4. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmoles/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmole/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Derived using calibration standard 4-methylumbelliferone (Sigma, Catalog # M1381).

  Per Well:
  • rXc beta -Galactosidase: 0.050 µg
  • Substrate: 200 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant X. campestris beta(1-3)-Galactosidase, CF

• beta (13)Galactosidase
• beta (1-3)-Galactosidase

Background

The majority of secreted and membrane proteins are glycosylated (1, 2). Proper glycosylation might be critical for protein folding and biological functions (3, 4). Galactoside is an essential sugar commonly found on various glycan conjugates and galactosidases are among the earliest enzymes to be studied (5). beta 1‑3 Galactosidase from Xanthomanas capestris is a useful tool for removing beta 1-3 linked galactosides from the non-reducing terminus of glycoconjugates (6, 7).

1. Lis, H. and Sharon, N. (1993) Eur. J. Biochem. 218:1.
2. Hart, G.W. (1992) Curr. Opin. Cell Biol. 4:1017.
3. Dwek, R.A. (1995) Biochem. Soc. Trans. 23:1.
4. Wormald, M.R. and Dwek, R.A. (1999) Structure 7:R155.
5. Hood, J.M. et al. (1977) Proc. Natl. Acad. Sci. USA 75:113.
6. Taron, C. et al. (1995) Glycobiology 5:603.
7. Glasgow, L. et al. (1977) J. Biol. Chem. 252:8615.

Bioinformatics

• Uniprot:
  • NP_638243()