The enzyme releases non-reducing terminal β(1-4)-linked galactose from oligosaccharides and glycoproteins. This specificity is only evident at enzyme concentrations < 100mU/ml. At higher concentrations, hydrolysis of β(1-3)-linked galactose occurs.
Figure 1: Cleavage specificity for GKX-5014 β(1-4)-Galactosidase.
Due to its high selectivity the enzyme is an extremely useful reagent for the identification of non-reducing terminal β(1-4)-linked galactose residues. As such the enzyme has been extensively used for detailed structural analysis in conjunction with broader specificity bovine testes β-galactosidase (GKX-5013) or Jack bean β-galactosidase (GKX-5012).
Suggestions for Use:
Procedure For De-galactosylation
1. Add up to 100 μg of asialoglycoprotein or 1 nmol of oligosaccharide to tube.
2. Add de-ionized water to a total of 14 μl.
3. Add 4 μl of 5x Reaction Buffer.
4. Add 2 μl β(1-4) Galactosidase.
5. Incubate at 37 °C for 1 hour.
For glycoproteins, cleavage may be monitored by SDS-PAGE if the size differential between native and de-galactosylated protein is sufficient for detection.
For the cleavage of Galβ(1-4) linkages occurring in isolated glycans, the substrate reaction concentration should be approximately 10-20 μM. Reconstitute the enzyme in 1X Incubation buffer to give a reaction concentration of 80 mU/mL. Incubate for up to 18 hours at 37 °C.
WS0049 5x Reaction Buffer B [250 mM sodium phosphate (pH 6.0)]
20 mM Tris-HCl, 25 mM NaCl (pH 7.5)
5X concentrated buffer which when diluted gives 50 mM sodium phosphate pH 6.0.
MW: 220-247 kD
One unit is defined as the amount of enzyme required to hydrolyze 1 μmole oNP-β-D-galactopyranoside per min at pH 6.0 and 37°C.
Size: 200 mU (100 µl)
Concentration: ≥ 2 U/ml
Product Code: GKX-5014
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Product Safety Documentation for GKX-5014: