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O glycanase

Manufactured by New England Biolabs

O-glycanase is an enzyme used to remove O-linked glycans from glycoproteins. It functions by cleaving the glycosidic linkage between the carbohydrate and the serine or threonine residue of the polypeptide backbone.

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2 protocols using o glycanase

1

Deglycosylation and Hydrolase Binding Assay of OMC45

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OMC45 polypeptide was treated with N-glycanase and O-glycanase separately following the manufacturer’s instructions. Both N-glycanase and O-glycanase were purchased from New England Biolabs., MA. After enzymatic digestion, deglycosylated OMC45 polypeptide was analzed by Western blot stained with anti-OMC45.
For the hydrolase binding experiments, the homogenous fraction of OMC45 polypeptide was first treated with N-glycanase (to remove all N-linked oligosaccharide moieties) and the resulting OMC45 polypeptide was treated with O-glycanase (an endoenzyme known to cleave O-linked OS chains). Both incubations were done overnight at 37°C according to the New England Biolabs’ instructions. After enzymatic digestion, deglycosylated OMC45 polypeptide and native OMC45 polypeptide were conjugated to AminoLink Plus coupling gel (Pierce Chemical Co.) separately at pH 10.0 according to the manufacturer’s instructions. As a control, same units of N-glycanase and O-glycanase were conjugated to AminoLink Plus coupling gel. A centrifugation assay was performed to determine the binding efficacy of hydrolases to the deglysosylated OMC45 polypeptide following the method of Nagdas et al. [13 (link)].
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2

O-Glycosidic Linkage Removal from Proteins

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To remove O-linked glycans from proteins, whole cell lysates in RIPA buffer were treated with Neuraminidase (New England Biolabs GmbH, Frankfurt am Main, Germany; Cat. No. P0720S) and Endo-α-N-acetylgalactosaminidase, also known as O-glycanase (New England Biolabs; Cat. No. P0733S), according to the manufacturer's instructions. Therefore, a total protein amount of 90 μg was adjusted with RIPA buffer to a final volume of 45 μl, followed by the addition of 5 μl of 10× Glycoprotein Denaturing Buffer (5% SDS, 0.4 M DTT). Proteins were denatured by heating at 95°C for 10 min. The chilled reaction was supplemented with 7 μl G7 Buffer (0.5 M sodium phosphate, pH 7.5) and 7 μl of 10% NP-40. The reaction was divided in to three equal parts. One third was supplemented with 2 μl ddH2O giving the negative control. The second third was supplemented with 1 μl ddH2O and 1 μl Neuraminidase (50 U/μl). The last third was supplemented with 1 μl Neuraminidase and 1 μl O-glycanase (40,000 U/μl-1). All reactions were incubated at 37°C for 3.5 h. The reactions were stopped by addition of protein sample buffer (Roti-Load 1; Carl Roth, Karlsruhe, Germany, K929.1).
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