Behind the formation and protection of microtubules RockefellerUniv
in 1× SDS sample buffer was loaded on a 10-well, 4–20% Tris-glycine precast gel with “wide wells” . A current of 150 V was applied until the sample migrated ∼1 cm into the gel. An ∼1 cm × 1 cm gel “plug” was cut, and it was further cut into ∼1-mm cubes. Proteins in the gel were reduced and alkylated and then digested with trypsin and lysC . The generated peptides were analyzed using an Orbitrap Fusion Lumos LC-MS/MS using a 70-min gradient on a pulled-emitter column.
150 μl of cell lysate at a concentration of 2 μg/ml was layered at the top of the gradient. The gradient was centrifuged at 55,000 rpm in a SW-55 rotor at 4°C for 3.5 h with minimum acceleration and no break. 16 × ∼220 µl fractions were manually collected by inserting a needle into the bottom of the gradient and drawing the sample up using a peristaltic pump. Fractions were denatured in 5× sample buffer and boiled at 95°C for 10 min.
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