| <b>Unit Definition</b> One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 10 µg of denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 μl (10 NEB units = 1 IUB milliunit). Unit Definition Assay | 10 µg of RNase B are denatured with 1X Glycoprotein Denaturing Buffer (0.5% SDS, 40 mM DTT) at 100°C for 10 minutes. After the addition of 1X GlycoBuffer 3, two-fold dilutions of Endo H f are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE. 1X Glycoprotein Denaturing Buffer 0.5% SDS 40 mM DTT <b>Reaction Conditions</b> 1X GlycoBuffer 3 Incubate at 37°C 1X GlycoBuffer 3 50 mM sodium acetate (pH 6 @ 25°C) <b>Storage Buffer</b> 20 mM Tris-HCl 50 mM NaCl 5 mM EDTA pH 7.5 @ 25°C <b>Heat Inactivation</b> 75°C for 10 minutes <b>Molecular Weight</b> Apparent: 70 kDa |
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