Coomassie blue cbb g 250

Proteins of wild type and ethe1-1 (9 DAP, light, dark) were extracted from 30 mg of pulverized seeds (pooled sample) by adding 150 μL extraction buffer [4% SDS (w/v), 125 mM TRIS, 20% glycerol (v/v)] and incubating at 60°C for 5 min. Another 150 μL of ddH₂O were added to the sample followed by centrifugation at 18000 × g for 10 min. The protein concentration of the supernatant was measured by using the Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Dreieich, Germany). To each sample (total protein 50 μg) 2-mercapto ethanol with a trace of bromophenol blue was added to a final concentration of 5%. SDS gel electrophoresis using a 1D glycine gel (stacking gel 4% acrylamide, separating gel 14% acrylamide) was performed according to Laemmli (1970) (link). The gel run was stopped before proteins entered the separation gel. The gel was fixed with 10% (v/v) acetate in 40% (v/v) methanol for 45 min and stained with Coomassie blue CBB G-250 (Merck, Darmstadt, Germany) for 30 min as described by Neuhoff et al. (1985 , 1990 (link)). Gel bands were cut using a scalpel and diced into 1.0–1.5 mm cubes. Carbamidomethylated followed by tryptic digestion and extraction of proteins was performed according to Klodmann et al. (2010) (link). Resulting peptides were resolved in 20 μL of 2% [v/v] ACN, 0.1% [v/v] formic acid (FA) prior to MS analysis.