Colloidal coomassie blue staining

MTs were prepared by polymerizing porcine tubulin (Cytoskeleton inc.) in general tubulin buffer (80mM PIPES pH 6.9, 2mM MgCl₂, 0.4mM EGTA, Roche Protease Inhibitors) in the presence of 1mM GTP for 20 min at 35C and then diluted further. To prevent depolymerization, MTs were treated with 40μM Taxol. MTs and 5μg purified protein were incubated at room temperature, and pelleted at 100,000g over a 60% glycerol cushion buffer (80mM PIPES pH 7.0, 1 mM MgCl2, 1 mM EGTA, 60% Glycerol, protease inhibitor). The supernatant (top layer above cushion) and the pellet were removed and treated with 4X Sample Buffer with 250mM DTT and 5% beta-mercaptoethanol. Resultant samples were subject to SDS-PAGE and colloidal Coomassie blue staining (Invitrogen).

Proteins were extracted and purified from ground samples using a standard phenol extraction method, then followed by ammonium acetate-methanol precipitation (Zhenget al., 2013 (link)). Concentrations in all extracts were determined with an RC/DC™ protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The proteins extracted from replicates 1–3 were pooled with the same quantity for proteomics biological replicate 1, whereas replicates 4–5 were for biological replicate 2. Further quantification was conducted on a pre-cast NOVEX 12% Tris/Glycine mini-gel (Invitrogen, Carlsbad, CA, USA) along with a series ofEscherichia coli lysates (2, 5, 10 or 20 µg per lane) (Supplementary Fig. S2). The SDS gel was visualized with colloidal Coomassie blue staining (Invitrogen), then imaged by a Typhoon 9400 scanner and Image Quant Software version 5.2 (GE Healthcare).