Amicon ultra 15 ml centrifugal filter devices

C. neoformans strains bim1∆ (DTY1000), bim1∆ complemented with either Bim1-HA (DTY1005), Bim1-HA (∆GPI) (DTY1007) and Bim1-HA H64A H65A (DTY1009) were grown in YPD agar lawns for 72 h (2 plates per strain). Cells were harvested and resuspended in secretion buffer (10 mM imidazole, 2% glucose, pH 5.0) (ratio of 5 ml/plate) and incubated at 30 °C for 17 h with shaking. After incubation, cells were pelleted and supernatants from 10 ml of overnight secretions (2 plates) were filtrated through 0.45 μm pore size filters (to facilitate supernatant concentration) and concentrated to approximately 200 μl (~ 6 μg/μl of total protein) in Amicon® Ultra 15 ml centrifugal filter devices (Millipore) with a 10,000 NMWL. After supernatant concentration, protein from all strains was normalized to 5 μg/μl in 200 μl of medium. 15 μl were used for immunoblotting analysis and the rest was diluted into 7 ml of SC medium. The 7 ml of medium, containing secreted proteins (including secreted Bim1-HA or mutants of Bim1-HA), were filter sterilized using 0.2 μm pore size filters. C. neoformans bim1∆ cells grown overnight in SC medium were diluted to an OD₆₀₀ of 0.004 in 2 ml of each of the filter sterilized SC medium containing the secreted proteins from the different strains. Quantitative liquid growth assays were performed as described below.