Tris hcl sds page

Yeast were grown in galactose-containing media to induce protein expression for 5h (for strains expressing Hsp104s alone or with TDP-43) or 8h (for strains expressing αSyn). Cultures were normalized to OD600=0.6, and 6 mL of cells were harvested. For heat shock controls, samples were incubated at 37 °C for 30 minutes prior to processing. Yeast lysates were extracted by incubation with 0.1M NaOH at room temperature for 5 min. Lysates were mixed with SDS sample buffer, boiled for 5 min, and subjected to Tris-HCl SDS-PAGE (4-20% gradient, Bio-Rad) followed by transfer to a PVDF membrane (Millipore).
Membranes were blocked in Odyssey blocking buffer (LI-COR) for 1h at room temperature or overnight at 4 °C. Primary antibody incubations were performed at 4 °C overnight or at room temperature for 2h. After washing with PBST, membranes were incubated with fluorescently labeled secondary antibodies at room temperature for 1h, followed by washing with PBST. Proteins were detected using an Odyssey Fc Dual-Mode Imaging system. Primary antibodies used: mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), rabbit polyclonal anti-TDP-43 (Proteintech), rabbit polyclonal anti-GFP (Sigma-Aldrich), mouse monoclonal 3-phosphoglycerate kinase (Novex), rabbit polyclonal anti-Hsp26 (Johannes Buchner, TU-Munich), fluorescently labeled anti-mouse and antirabbit secondary antibodies (Li-Cor).