Simplyblue coomassie

Immunoisolation samples were loaded on 4–12% Bis-Tris gradient gels and stained with Simply Blue Coomassie (Invitrogen, Carlsbad, California, USA). The gel bands from the IgG or EAAT2 immunoisolation were excised above 50 kD and excess stain was removed by an overnight wash of 50% 100 mM ammonium bicarbonate/50% acetonitrile. After destaining, disulfide bonds were reduced by 25 mM dithiothreitol at 50 °C for 30 min. Alkylation of the free thiol groups was carried out with 55 mM iodoacetamide for 30 min in the dark. The excess alkylating agent was removed and the gel pieces were washed twice with a 100 mM ammonium bicarbonate for 30 min. The gel pieces were evaporated to dryness in a SpeedVac (Savant) before the addition of the enzyme. 12.5 ng/μl of trypsin (Promega Gold Mass Spectrometry Grade) was added to each gel sample and incubated overnight at 37 °C. Peptides were extracted from the gel pieces using a 1:1 mixture of 5% formic acid and 50% aqueous acetonitrile twice for 15 min. Extracts were pooled and evaporated to dryness. The samples were then resuspended in 20 μl of a 0.1% formic acid prior to mass spectrometry analysis.