Recombinant X. campestris beta(1-3)-Galactosidase, CF Summary
Ser25-Glu613 with an N-terminal Met and 6-His tag
Accession # NP_638243
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.
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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 0.1 M MES, pH 5.5
- Recombinant X. campestris beta (1‑3)-Galactosidase (rXc beta -Galactosidase) (Catalog # 5704-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 rXc beta -Galactosidase to 1 ng/µL in Assay Buffer.
- Dilute Substrate to 400 µM in Assay Buffer.
- Load into plate 50 µL of 1 ng/µL rXc beta -Galactosidase, and start the reaction by adding 50 µL of 400 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL 400 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 (pmoles/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmole/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard 4-methylumbelliferone (Sigma, Catalog # M1381).Per Well:
- rXc beta -Galactosidase: 0.050 µg
- Substrate: 200 µM
Background: beta (1-3)-Galactosidase
The majority of secreted and membrane proteins are glycosylated (1, 2). Proper glycosylation might be critical for protein folding and biological functions (3, 4). Galactoside is an essential sugar commonly found on various glycan conjugates and galactosidases are among the earliest enzymes to be studied (5). beta 1‑3 Galactosidase from Xanthomanas capestris is a useful tool for removing beta 1-3 linked galactosides from the non-reducing terminus of glycoconjugates (6, 7).
- Lis, H. and Sharon, N. (1993) Eur. J. Biochem. 218:1.
- Hart, G.W. (1992) Curr. Opin. Cell Biol. 4:1017.
- 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.
- Taron, C. et al. (1995) Glycobiology 5:603.
- Glasgow, L. et al. (1977) J. Biol. Chem. 252:8615.
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