Coomassie blue g 250 stain

For fast liquid protein chromatograph (FPLC) 1–2 mg of protein was dissolved in 500 μl of DI water with 50 mM NaCl, adjusted to pH 2 with HCl, as running buffer. Samples were injected into a Superdex 75 10/300 GL column (GE Healthcare) mounted on an ATKA PURE (GE Healthcare) FPLC. The column was typically rinsed with 2 column volumes of DI water, and then washed with running buffer for one column volume before injection of the sample. For separation, UV absorption was monitored at 280 nm during column filtration and samples were fractionated into test tubes.
For reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), samples of fresh monomers, early-stage oligomers ESOs) and late-stage oligomers (LSOs) were prepared and isolated as described above. All samples were analyzed using 16.5% gradient Tris-tricine gels (Criterion, Bio-Rad) with an SDS running buffer without glycine. An aliquot of each sample (2 mg ml⁻¹) was mixed with Tricine sample buffer (Bio-Rad) in the presence of 2% (v/v) 2-mercaptoethanol (Fisher Scientific) in 1 : 1 ratio and boiled for 5 minutes at 90 °C. 30 μl of each sample was loaded onto the gel and run for about 1.5 hours at 105 V. The gel was fixed in a solution of 40% methanol and 10% acetic acid for 30 minutes, stained for 2 hours using Coomassie blue (G-250 stain, Bio-Rad) for 1 hour, and was washed overnight with DI water.