Rabbit igg agarose

The selected strains were grown to mid-log exponential phase (OD₆₆₀ = 1) and were left unstressed or were stressed as described above except for oxidative stress where cells were stressed in 2 mM H₂O₂. The cells were then collected (400 ml per condition) and kept at −80 °C. Cell pellets were resuspended in 2 ml of lysis buffer (45 mM Hepes-KOH at pH 7.2, 150 mM NaCl, 1 mM EDTA, 10% glycerol, and 1% Triton X-100) containing a cocktail of protease and phosphatase inhibitors. An equal volume of glass beads (0.5-mm diameter) was added, and the cells were broken by vortexing at 4 °C. The whole extract was clarified by centrifugation for 10 min at 9300 × g at 4 °C and an aliquot was taken as the whole-cell extract (WCE). The extracts (3–7 mg) were first incubated for 3 h with 1:100 dilutions of anti-Myc antibody (9E10) or with anti-HA antibody (12CA5) and were subsequently incubated overnight with the protein G affinity matrix (GE Healthcare). For purification of the TAP-tagged proteins, cell extracts were directly incubated with rabbit IgG–Agarose (Sigma) overnight at 4 °C. The agarose beads were then washed 10 times with the lysis buffer. Antibody-bound fractions and the corresponding WCE were boiled in SDS-containing sample buffer and were analyzed using 8% SDS-PAGE. TAP-tagged proteins were detected with a 1:5 dilution of anti-PAP antibody (p1291 from Sigma).

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.

TAP-tagged Spt4 and Spt4^T42AS43A cells were grown to mid-log phase on YPD and were then subjected to a brief osmostress (0.4 M NaCl for 5 minutes) or were left untreated. For each condition, 100 ml aliquots were harvested by centrifugation and pellets were resuspended in 300 μl of buffer A (50 mM Tris-HCl pH 8.0, 15 mM EDTA, 15 mM EGTA, 0.1% Triton X-100 ± 150 mM NaCl) supplemented with protease and phosphatase inhibitors (1 mM PMSF, 2 μg ml^-1 leupeptin, 2 μg ml^-1 pepstatin, 1 mM benzamidine, 2 mM DTT, 10 mM sodium fluoride, 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate). Chilled glass beads were added, and cells were disrupted with a Fast-prep glass bead beater (Millipore). Protein extracts (10 mg) were incubated with rabbit IgG agarose (Sigma) beads for 2–3 hours at 4°C. Beads were washed 10 times with supplemented buffer A + NaCl, and once with 50 mM Tris-HCl pH 6.8, boiled in SDS-loading buffer for 5 minutes and analyzed by 11% SDS-PAGE. Proteins were then transferred to PVDF membranes and phosphorylation was detected by immunoblotting with an anti-phospho Ser/Thr antibody (BD Transduction Laboratories).