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Aminolink plus coupling gel

Manufactured by Thermo Fisher Scientific

AminoLink Plus coupling gel is a silica-based, activated resin designed for the covalent immobilization of ligands containing primary amine groups. It provides a simple and effective method for coupling a wide range of amine-containing biomolecules, including proteins, peptides, and small molecules, to a solid support.

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2 protocols using aminolink plus coupling gel

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

Proteolytic Activation of Mutant TRACPs

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To examine if mutant intracellular TRACPs could be activated by proteolysis in vitro from isoform 5a-like to 5b-like enzymes, TRACP-HEK lysates in 2 μl WT cells or 10 μl culture medium were digested with 200 mU solid-phase trypsin-agarose (Sigma Chem Co, St. Louis, MO) or control BSA-conjugated agarose (2 mg BSA /ml packed agarose) made with Amino Link Plus coupling gel (Pierce Chemical Co., Rockford, IL) for 2h at room temperature. Reactions were made to 50 μl with 10 mM Tris / 30 mM NaCl / 0.1% Triton X-100, pH 7.0. After incubation, the solid-phase trypsin and BSA were removed by centrifugation. Digests were subjected to Western blot analysis with Mab9C5.
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