Chaps

C. neoformans H99 (serotype A) and 1841 (serotype D) cells were recovered from 10% skim milk stocks stored at -80°C and grown independently for 48 h in Sabouraud dextrose broth while shaking (80 rpm) at 30°C. Cells were harvested by centrifugation and washed twice with 250 mM sucrose. The pellet was resuspended in lysis buffer [5 mM Tris/HCl pH 7.5, 2.5 mM EDTA, 0.5X protease inhibitor cocktail (Roche, Basel, Switzerland)] additionally containing 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, Cat. No. 1479, Carl Roth, Karlsruhe, Germany) and 50 mM dithiothreitol (DTT, Cat. No. 6908, Carl Roth, Germany). The cell suspension was transferred into a mortar, frozen with liquid nitrogen and homogenized with a pestle twice. Afterwards, homogenates were centrifuged and supernatant was recovered. Protein content was estimated using Bradford reagent (Carl Roth, Karlsruhe, Germany). Proteins were precipitated with 10% trichloroacetic acid over night at -20°C and centrifuged. After removal of the supernatant, the pellet was washed three times with ice-cold acetone and air-dried. The protein pellet was dissolved in a solution containing 7 M urea, 2 M thiourea, and 4% CHAPS and protein content was estimated using Bradford reagent (Carl Roth, Germany).

Tris, urea, thiourea, CHAPS, dithiothreitol, bromophenol blue, glycerin, sodium dodecyl sulfate, glycine, temed, ammoniumperoxodisulfate, ammonium sulfate, ammonium bicarbonate, colloidal Coomassie Blue, and acrylamide were purchased from Roth (Karlsruhe, Germany). Iodacetamide was obtained from SERVA (Heidelberg, Germany) and benzonase was purchased from Novagen (Darmstadt, Germany). Ampholytes (Biolyte 3–10) were purchased from Bio-Rad Laboratories (München, Germany) and DeStreak was obtained from Amersham Bioscience (Freiburg, Germany).

Protein preparation was performed at 4°C. LCL pellets were resuspended in 750 μl DIGE lysis buffer (7 M urea, 2 M thiourea, 30 mM Tris pH 8.5 and 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), all from Roth, Karlsruhe, Germany) and subjected to five successive freeze and thaw cycles. Yeast cell pellets were resuspended in 1 ml DIGE lysis buffer for yeast (9 M urea, 4% CHAPS, 30 mM Tris pH 8.8, all from Roth) and mechanically lysed using 0.45 mm acid-washed glass beads. The cytosolic protein fractions from LCLs or yeast cells were purified by centrifugation at 13,000 × g for 20 minutes. The protein concentration of the supernatant was measured using the Protein Bradford Assay (Biorad, Munich, Germany).

2D electrophoresis was performed essentially as described previously [12] (link). In brief, Immobiline DryStrips (24 cm, 3–11 NL, Amersham, Freiburg, Germany) were rehydrated with 2D electrophoresis buffer (7 M urea (Roth, Karlsruhe, Germany), 2 M thiourea (Sigma-Aldrich, Steinheim, Germany), 4% [w/v] CHAPS (Roth) 1% [v/v] IPG buffer 3–11 NL) supplemented with 1.2% [v/v] DeStreak reagent (Amersham). Extracts of dEAC_intens (2×400 µg) were precipitated with acetone and pellets were dissolved in 2D electrophoresis buffer supplemented with 1% [w/v] DTT. Isoelectric focusing was performed in an Ettan IPGphor (Amersham) using anodal cup loading. For the second dimension, IPG strips were equilibrated twice with equilibration buffer (100 mM Tris-HCl pH 8.0, 6 M urea, 30% [v/v] glycerol (Roth), 2% [w/v] SDS (Serva, Heidelberg, Germany) supplemented with 0.5% [w/v] DTT for the first equilibration step and 4.5% [w/v] iodoacetamide (Sigma-Aldrich) for the second step. The second dimension was performed with 12.5% polyacrylamide gels applying the Ettan DALTsix electrophoresis system (Amersham). Two identical experiments were performed and the gels were either stained with silver [13] (link) or subjected to western blotting.