Conjugation of anti-isopeptide monoclonal to Dynabeads-Protein G

Mouse anti-isopeptide monoclonal 81D1C2 was conjugated to Dynabeads-Protein G using the Sporty et al. [29] protocol. A 100 μL suspension of Dynabeads-Protein G was transferred to a microfuge tube, where the magnetic beads were washed 3 times with phosphate buffered saline (PBS). A 20 μg aliquot (20 μL) of antibody and 380 μL of PBS were added to the washed 3 mg beads to make a 0.05 mg/mL antibody solution. The sample was rotated overnight at room temperature to allow the antibody to bind to Protein G. The supernatant was discarded and the beads were washed 2 times with 0.2 M triethanolamine hydrochloride pH 7.8, 0.02% (w/v) sodium azide. A freshly prepared 200 μL solution of 0.010 g dimethyl pimelimidate dihydrochloride in 1.8 mL of 0.2 M triethanolamine pH 7.8, 0.02% azide was added to the beads. The tube was rotated 30 min at room temperature. After 30 min, the liquid was discarded. The conjugation reaction was quenched by incubating the beads with 200 μL of 20 mM TrisCl, 0.15 M NaCl for 15 min. The beads were washed 3 times with 200 μL of 20 mM TrisCl, 0.15 M NaCl containing 0.05% (v/v) of Tween-20. The washed beads were used immediately for immunopurification of isopeptides.

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