Colloidal coomassie blue

Sup35-TAP affinity purification was performed as described previously [37 (link)]. Sup35-interacting proteins were identified in the wild-type and tsa1 tsa2 mutant by mass spectrometry in triplicate for each strain. For protein identification, protein samples were run a short distance into SDS-PAGE gels and stained using colloidal Coomassie blue (Sigma). Total proteins were excised, trypsin digested, and identified using liquid chromatography-mass spectrometry (LC-MS) performed by the Biomolecular Analysis Core Facility, Faculty of Biology, Medicine and Health, University of Manchester. Proteins were identified using the Mascot mass fingerprinting programme (www.matrixscience.com) to search the NCBInr and Swissprot databases. Final datasets for each condition were determined by selecting proteins that were identified in at least two of the three replicates.

Yeast cells (BY4742 strain background) were grown to exponential phase (A₆₀₀ ∼0.6) in YPD medium without or with arsenite (1.5 mM sodium arsenite, 1 hour) and equivalent cell numbers (10 A₆₀₀ units) were used to isolate aggregated proteins as described previously (Jacobson et al., 2012 (link); Rand and Grant, 2006 (link)). Briefly, cells were disrupted in lysis buffer (50 mM potassium phosphate buffer, pH 7, 1 mM EDTA, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride and Complete Mini protease inhibitor cocktail (Roche)), and membrane proteins and aggregated proteins were isolated by centrifugation (15,000 g; 20 minutes). Membrane proteins were removed by washing twice with 320 µl lysis buffer and 80 µl of 10% Igepal CA 630 (NP-40) (Sigma–Aldrich), centrifuging at 15,000 g for 20 minutes each time, and the final aggregated protein extract was resuspended in 100 µl of lysis buffer. Aggregated proteins were separated on 12% reducing SDS-PAGE gels and stained using colloidal Coomassie blue (Sigma–Aldrich). Proteins were excised, trypsin-digested, and identified using liquid chromatography-mass spectrometry (LC-MS) in the Biomolecular Analysis Facility (Faculty of Life Sciences, University of Manchester). Proteins were identified using the Mascot mass fingerprinting programme (http://www.matrixscience.com) to search the NCBInr and Swissprot databases.

Following SD (untreated), AZC (5 mM) or H₂O₂ (1 mM) treatment for 2 hours, insoluble protein aggregates were isolated as previously described16 (link)22 (link). Insoluble protein extracts were separated by reducing SDS/PAGE (12% gels) and visualized by silver staining with the Bio-Rad silver stain plus kit. Aggregated proteins were identified by mass spectrometry (performed by the Biomolecular Analysis Core Facility, Faculty of Life Sciences, The University of Manchester) in triplicate for each condition. For protein identification, protein samples were run a short distance into SDS-PAGE gels and stained using colloidal Coomassie blue (Sigma). Total proteins were excised, trypsin digested, and identified using liquid chromatography-mass spectrometry (LC-MS). Proteins were identified using the Mascot mass fingerprinting programme (www.matrixscience.com) to search the NCBInr and Swissprot databases. Final datasets for each condition were determined by selecting proteins that were identified in at least two of the three replicates.