Ab186695

Yeast strains were grown in standard synthetic dextrose (SD) medium (6.7 g/L yeast nitrogen base with ammonium sulfate, 2% galactose) from OD600 of~0.1 overnight at 30°c. Next, the cultures were back diluted to OD600 of~0.1 and let to grow until OD600 of~0.5. The media fraction was separated from the cells using centrifugation (3000g for 3min) and cells were washed with DDW. Both media and cell fractions were precipitated using 10% Trichloroacetic acid (TCA) (Sigma) for 20min on ice, centrifuged for 15min at 14000g at 4°c, the supernatant was aspirated, pellet was washed in cold acetone, dried at room temperature for 30min and resuspended in urea lysis buffer (8M urea in 50mM tris pH 7.5 and oComplete Protease Inhibitor (Roche)). The cells were beaten with 100μl of glass beads (scientific industries) for 10min at 4°c. Then 0.1% SDS and 50mM DTT were added to both the cells and the media fractions and boiled at 95°c for 5min. Glass beads and cell debris were removed and the samples were resolved on 4-20% precast polyacrylamide gel (Bio-Rad), transferred to nitrocellulose membrane (PALL), and probed with a monoclonal mouse α-cherry (ab125096, Abcam) and a polyclonal rabbit α-GFP (ab290, Abcam). Secondary antibodies were alexa680 α-rabbit (ab175773, Abcam) and alexa790 α-mouse (ab186695, Abcam) that enable scanning using an Odyssey imaging system (LI-COR Biosciences).