Easytag express

To measure the expression of FLAG-tagged proteins under iron starvation conditions, C. albicans cells were grown overnight in IPM at 30°C, diluted 1∶20 in the same medium, and grown 2 hours to OD₆₀₀ = 1.3. 1.5 ml of the culture were spun down, washed twice in low-iron YNB +1 mM ferrozine, resuspended in 0.1 ml of this medium supplemented with 0.25 mCi ³⁵S methionine/cysteine (Perkin Elmer Easytag Express) and incubated for 5 min at 30°C. Proteins were then extracted and immunoprecipitated as described in [60] (link), using a rabbit anti-FLAG antiserum Proteins were separated by SDS-PAGE and visualized and quantitated using a GE Typhoon FLA 7000 phosphorimager.

Exponentially growing pADH-HA-ADE2 or pADH-HA-43GAr-ADE2 yeast cells were pulse-labeled with 90 μCi/OD₆₀₀ (EasyTag Express protein labeling mix [³⁵S], PerkinElmer Life Sciences) for 15 minutes after being cultured in methionine-free medium for 15 minutes, and then chased in fresh medium containing 50 mM cold methionine and cysteine for the indicated time points before harvesting. After centrifugation, cell pellets were resuspended in lysis buffer [25 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.2% Triton X-100, antiprotease cocktail (Roche), 1 mM phenyl-methylsulfonyl fluoride] and treated as described above. Lysates were pre-cleared with protein G-Sepharose beads for 45 minutes at 4°C and further immunoprecipitated with 1 μg of anti-HA monoclonal antibodies pre-bound to protein G-Sepharose beads overnight at 4°C. The beads were then washed with PBS 1×/0.2% Igepal four times and boiled in SDS loading buffer. Immunoprecipitates were analyzed by SDS-PAGE using 10% precast NUPAGE gels (Invitrogen). The gel was dried and analyzed using a Typhoon 9400 Phosphorimager (GE).