Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to cleave a fluorogenic substrate, 4-Methylumbelliferyl-beta -D-galactopyranoside. The specific activity is >600 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .
Source
E. coli-derived Thr2-Leu595, with an N-terminal Met and a 6-His tag
Accession #
N-terminal Sequence
AnalysisMet
Predicted Molecular Mass
70 kDa
SDS-PAGE
62 kDa, reducing conditions
5549-GH |
| |
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. | ||
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.
|
Assay Procedure
Materials
Assay Buffer: 50 mM Sodium Phosphate, pH 6.0
Recombinant S. pneumoniae beta (1‑4)-Galactosidase (rSp beta (1-4)-Galactosidase) (Catalog # 5549-GH)
Substrate: 4-methylumbelliferyl-beta -D-galactopyranoside (Sigma, Catalog # M1633), 10 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rSp beta (1-4)-Galactosidase to 4 ng/µL in Assay Buffer.
Dilute Substrate to 500 µM in Assay Buffer.
Load into plate 50 µL of 4 ng/µL rSp beta (1-4)-Galactosidase and start the reaction by adding 50 µL of 500 µM Substrate. Include a Substrate Control containing 50 µL Assay Buffer, 50 µL of 500 µM Substrate.
Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), 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 (Sigma, Catalog # M1381).
Per Well:
rSp beta (1-4)-Galactosidase: 0.20 µg
Substrate: 250 µM
Background: beta (1-4)-Galactosidase
Majority of secreted and membrane proteins are glycosylated (1, 2). Proper glycosylation could be critical for protein folding and biological functions (3, 4). Galactosides are commonly found on various glycan conjugates and galactosidases are tools for removing these sugar residues (5). beta 1-4 Galactosidase from Streptococcus pneumoniae is especially efficient in removing beta 1-4 linked non-reducing galactosides from Gal-beta 1-4-GlcNAc or Gal-beta 1-4-GalNAc linkage (6).
References:
Lis, H. and Sharon, N. (1993) Eur. J. Biochem. 218:1.
Hart, G.W. (1992) Curr. Opin. Cell Biol., 4:1017.3.
Dwek, R.A. (1995) Biochem. Soc. Trans. 23:1.
Wormald, M.R. and Dwek, R.A. (1999) Structure 7:R155.
Hood, J.M. et al. (1977) Proc. Natl. Acad. Sci. USA 75:113.
Glasgow, L. et al. (1977) J. Biol. Chem. 252:8615.
Alternate Names:
beta (14)Galactosidase; beta (1-4)-Galactosidase; beta-Galactosidase-3
