详细说明
- 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.
- ActivityMeasured 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 .
- SourceE. coli-derived Val2-Val708, with an N-terminal Met and 6-His tag
- Accession #
- N-terminal Sequence
AnalysisMet - Predicted Molecular Mass82 kDa
- SDS-PAGE70-75 kDa, reducing conditions
| 6415-GH | | |
| 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).
- 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
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