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
AnalysisMet
Predicted Molecular Mass
82 kDa
SDS-PAGE
70-75 kDa, reducing conditions
6415-GH |
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Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. | ||
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.
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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
Dilute rE. coli alpha -Gal to 0.5 ng/μL in Assay Buffer.
Dilute Substrate to 800 μM in Assay Buffer.
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.
Read plate at excitation and emission wavelengths of 365 nm and 445 nm, respectively, 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 (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:
Schmid, K. and Schmitt, R. (1976) Eur. J. Biochem. 67:95.
Spangenberg, P. et al. (2000) Carbohydr. Res. 329:65.
Entrez Gene IDs:
5589615 (E. coli)
Alternate Names:
alphaGalactosidase; alpha-Galactosidase; rafA
