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
Paulson JC, Prieels JP, Glasgow LR, Hill RL. Sialyl- and fucosyltransferases in the biosynthesis of asparaginyl-linked oligosaccharides in glycoproteins. Mutually exclusive glycosylation by beta-galactoside alpha2 goes to 6 sialyltransferase and N-acetylglucosaminide alpha1 goes to 3 fucosyltransferase. J Biol Chem. 1978 Aug 25;253(16):5617-24.
Q. Can I use this enzyme in a digestion with multiple glycosidases?
A. We use an ammonium acetate buffer when we run multi-enzyme exoglycosidase digests that include GKX-5014 β(1-4)-Galactosidase. We suggest a 10X reaction buffer of 500mM ammonium acetate pH 5.5 w/ 0.05% azide. The reaction buffer can also be used diluted to 20X (25mM ammonium acetate) and lower with purified glycans. This buffer works with most of our exoglycosidases in an overnight digestion: GK80040, GK80021, GKX-5014, GKX-5013, GKX-5023, GKX-5007, GKX-5010. Generally, 2 μl of each exoglycosidase is used in a 20μl reaction with an overnight incubation (16 hours) at 37°C. Please Contact Us for more details.
If the glycans need to purified from the reaction prior to analysis by e.g. LC-MS, samples can be spun through Nanosep® 10K Omega spin filters to remove protein. For data using these methods, please see our poster, Characterizing Low-Abundance Glycans on Therapeutic Proteins.
Also, acetate buffers work better than phosphate for injecting onto LC (phosphate phase separates in acetonitrile), and acetate is volatile so samples can be dried down if necessary.
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Product Safety Documentation for GKX-5014: