| <b>Unit Definition</b> One unit is defined as the amount of enzyme required to cleave > 95% of the terminal α-D- N -acetylgalactosamine from 1 nmol (GalNAcα1-3)(Fucα1-2)Galβ1-4Glc-7-amino-4-methyl-coumarin (AMC), in 1 hour at 37°C in a total reaction volume of 10 μl. <b>Reaction Conditions</b> 1X GlycoBuffer 1 Supplement with 100 µg/ml Recombinant Albumin (Low-glycerol) Incubate at 37°C 1X GlycoBuffer 1 5 mM CaCl2 50 mM sodium acetate (pH 5.5 @ 25°C) <b>Storage Buffer</b> 20 mM Tris-HCl 50 mM NaCl 1 mM EDTA pH 7.5 @ 25°C <b>Heat Inactivation</b> 65°C for 10 minutes <b>Molecular Weight</b> Apparent | 47 kDa <b>Unit Assay Conditions</b> Two fold dilutions of α- N -Acetylgalactosamininidase are incubated with 1 nmol AMC-labeled substrate in 1X GlycoBuffer 1, supplemented with 100 µg/ml rHSA, in a 10 μl reaction. The reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized via thin layer chromatography (1). |
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