Incubation of MAP-rich tubulin with 100 μM chlorpyrifos oxon

A 0.2 mL solution of 0.5 mg/mL MAP-rich tubulin in 20 mM TrisCl pH 8.5, 0.01% azide was incubated with 2 μL of 10 mM chlorpyrifos oxon (dissolved in acetonitrile) to give a final concentration of 100 μM chlorpyrifos oxon. After 48 hours at 37˚C, the sample was diluted to 1 mL with 20 mM ammonium bicarbonate pH 8 and injected into a hydrated Slide-A-Lyzer Dialysis Cassette #66370 Thermo Scientific 7,000 MWCO. The sample was dialyzed against 2 x 450 mL of 20 mM ammonium bicarbonate pH 8 at 4˚C to remove excess chlorpyrifos oxon. The dialyzed sample was concentrated to 50 μL in a vacuum centrifuge before 70 μg were loaded on an SDS gel. Coomassie blue stained bands were cut out of the gel. Proteins extracted from gel slices were reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin as described [28] in preparation for liquid chromatography tandem mass spectrometry.

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