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Recombinant S. pneumoniae beta(1-4)-Galactosidase, CF

Catalog #: 5549-GH Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
5549-GH-100
R&D Systems Recombinant Proteins and Enzymes
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Recombinant S. pneumoniae beta(1-4)-Galactosidase, CF Summary

Product Specifications

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.
Source
E. coli-derived s. pneumoniae beta (1-4)-Galactosidase protein
Thr2-Leu595, with an N-terminal Met and a 6-His tag
Accession #
YP_815516
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
70 kDa
SDS-PAGE
62 kDa, reducing conditions

Product Datasheets

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5549-GH

Product Datasheet
COA
SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

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.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

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
  1. Dilute rSp beta (1-4)-Galactosidase to 4 ng/µL in Assay Buffer.
  2. Dilute Substrate to 500 µM in Assay Buffer.
  3. 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.
  4. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), 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 (Sigma, Catalog # M1381).

Per Well:
  • rSp beta (1-4)-Galactosidase: 0.20 µg
  • Substrate: 250 µM
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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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
  1. Lis, H. and Sharon, N. (1993) Eur. J. Biochem. 218:1.
  2. Hart, G.W. (1992) Curr. Opin. Cell Biol., 4:1017.3.
  3. Dwek, R.A. (1995) Biochem. Soc. Trans. 23:1.
  4. Wormald, M.R. and Dwek, R.A. (1999) Structure 7:R155.
  5. Hood, J.M. et al. (1977) Proc. Natl. Acad. Sci. USA 75:113.
  6. Glasgow, L. et al. (1977) J. Biol. Chem. 252:8615.
Alternate Names
beta (14)Galactosidase; beta (1-4)-Galactosidase; beta-Galactosidase-3

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