Tandem Affinity Purification of Candida albicans Proteins

These were performed as described previously [20 (link)]. Briefly, C. albicans cultures were grown to OD₆₀₀ = 0.5, cells harvested, washed with sterile distilled H₂O and resuspended in 1 ml lysis buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl₂, 20% glycerol, 1 mM PMSF (EMD Chemicals), 20 mM Na₂MoO₄ (Sigma), and complete EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics). Cells were then disrupted by bead beating twice for 4 minutes with 7 minute breaks on ice between cycles. Lysates were centrifuged at 13,006 g for two-times 5-minutes, recovering the supernatants at each stage. The combined lysate was then cleared by centrifugation at 21,006 g for 10 minutes at 4°C and protein concentrations determined using the Bradford assay. Anti-TAP immunoprecipitations were performed by adjusting protein concentration to 1.5 mg/ml in lysis buffer containing 0.2% Tween, and incubating with Rabbit IgG agarose (Sigma #A2909) at 4°C overnight as per the manufacturer’s specifications. Unbound material was discarded, the beads were washed five times with 1 ml lysis buffer containing 0.1% Tween, and proteins were eluted by boiling in one volume of 2.5% sample buffer (125 mM Tris-HCl, pH 6.8, 5% glycerol, 2.5% SDS, 2.5% beta-mercaptoethanol, distilled H₂O, bromophenol blue). Proteins were separated on 8% SDS-PAGE gels and probed as above.