Recombinant E. coli alpha-Galactosidase/a-Gal Protein, CF

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

Recombinant E. coli alpha-Galactosidase/a-Gal Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze 4-methylumbelliferyl-alpha -D-galactopyranoside. The specific activity is >3,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived e. coli alpha-Galactosidase/a-Gal protein
  Val2-Val708, with an N-terminal Met and 6-His tag
• Accession #: YP_001464379
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: rafA
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 82 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: 70-75 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 100 mM MES, pH 6.5
  • Recombinant E. coli alpha ‑Galactosidase/ alpha ‑Gal (rE. coli alpha -Gal) (Catalog # 6415-GH)
  • Substrate: 4-Methylumbelliferyl-alpha -D-galactopyranoside (Sigma, Catalog # M7633), 6.7 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 rE. coli alpha -Gal to 0.5 ng/μL in Assay Buffer.
  2. Dilute Substrate to 800 μM in Assay Buffer.
  3. Load into a plate 50 μL of 0.5 ng/μL rE. coli alpha -Gal, and start the reaction by adding 50 μL of 800 μM Substrate. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of 800 μM Substrate.
  4. Read plate at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in kinetic mode for 5 minutes.
  5. 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 (4-MU) (Sigma, Catalog # M1381).

  Per Well:
  • rE. coli alpha -Gal: 0.025 μg
  • Substrate: 400 μM

Notes

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

Alternate Names for Recombinant E. coli alpha-Galactosidase/a-Gal Protein, CF

• agalsidase alfa
• Agalsidase alpha
• Agalsidase
• Alpha-D-galactosidase A
• alpha-D-galactoside galactohydrolase 1
• Alpha-D-galactoside galactohydrolase
• alpha-gal A
• alpha-galactosidase A
• EC 3.2.1
• EC 3.2.1.22
• GALA
• galactosidase, alpha
• GLA
• Melibiase

Background

alpha -Galactoside is prevalent in animals and plants. The alpha -Galactosidase ( alpha -Gal) from Escherichia coli is a useful tool for the removal of alpha 1,3-linked and alpha 1,6‑linked galactosides from the non-reducing terminus of complex carbohydrates and glycoproteins (1). alpha -Gal efficiently hydrolyzes raffinose, an alpha -D-galactosylsucrose, to D‑galactose and sucrose. The enzyme can also hydrolyze other alpha -Galactosides such as melibiose, stachyose, verbascose, and galactinol. The enzyme does not cleave beta -linked galactose, such as lactose.

1. Schmid, K. and Schmitt, R. (1976) Eur. J. Biochem. 67:95.
2. Spangenberg, P. et al. (2000) Carbohydr. Res. 329:65.

Bioinformatics

• Gene Symbol: rafA
• Uniprot:
  • YP_001464379()