Hydrolyzes non-reducing terminal galactose β(1-3) and β(1-4) linkages. Can be used in conjunction with other β-galactosidases for exoglycosidase sequencing.
Figure 1: Cleavage specificity for GKX-5013 β(1-3,4)-Galactosidase.
The enzyme has applications in the analysis of a wide variety of glycoconjugates. It is particularly useful for ensuring the complete removal of β(1-3) and β(1-4)-linked non-reducing terminal galactose residues of oligosaccharides. Gal β(1-6) GlcNAc is hydrolyzed more slowly, however this linkage is not normally encountered in native complex glycans. This activity towards β(1-3) and β(1-4)-linked galactose contrasts with that of our other β-galactosidases (e.g. from S.pneumoniae, GKX-5014, or Jack bean, GKX-5012) which exhibit a preference for Gal β(1-4), and cleave the Gal β(1-3) linkage relatively slowly, if at all. Used in conjunction, these enzymes provide a powerful means to determine linkage positions of non-reducing β galactose residues.
WS0063 5x Reaction Buffer [500 mM sodium citrate/phosphate pH 4.0]
20 mM sodium citrate phosphate, 150 mM NaCl (pH 4.0)
5X concentrated buffer which when diluted gives 100 mM sodium citrate/phosphate pH 4.0.
MW: ~68 kD
One unit is defined as the amount of enzyme required to hydrolyze 1 μmole of pNP-β-D-galactopyranoside per minute at pH 4.0 and 37°C.
Size: 0.5 U (100 μl)
Concentration: ≥ 5 U/ml
Product Code: GKX-5013
Q. What are the suggested reaction conditions for the enzyme?
A. For the digestion of isolated glycans and glycoproteins, the enzyme can be used at a concentration of around 0.5-2 U/ml in reaction buffer between pH 4.0-5.0. In general, 100 mU of enzyme in a final volume of 50-100 μl is normally sufficient to de-galactosylate 1-10 mg of glycoprotein following incubation at 37°C for 18 hours.
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-5013 β(1-3,4)-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.
Product Safety Documentation for GKX-5013: