• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  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. See Activity Assay Protocol on .

• Source

  E. coli-derived Val2-Val708, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met  

• Predicted Molecular Mass

  82 kDa

• SDS-PAGE

  70-75 kDa, reducing conditions

Assay Procedure

Materials

• 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:

     *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

Background: alpha-Galactosidase/a-Gal

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.

• References:

1. Schmid, K. and Schmitt, R. (1976) Eur. J. Biochem. 67:95.

2. Spangenberg, P. et al. (2000) Carbohydr. Res. 329:65.

• Entrez Gene IDs:

  5589615 (E. coli)

• Alternate Names:

  alphaGalactosidase; alpha-Galactosidase; rafA