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

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Product Details

Summary
Reactivity EcSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

Order Details

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 #
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

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

  • alphaGalactosidase
  • alpha-Galactosidase
  • rafA

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.

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Bioinformatics

Gene Symbol rafA
Uniprot