Hyglo development solution

Yeast were cultured to log phase (A₆₀₀ 0.75–1.0) in selective SC-uracil at 25° unless otherwise noted. Cell pellets of equal mass were harvested by centrifugation, washed with water, and processed by alkaline hydrolysis and trichloroacetic acid precipitation (Kim et al. 2005 (link)). Total cell precipitates were resuspended in urea-containing Sample Buffer (250 mM Tris, 6 M urea, 5% β-mercaptoethanol, 4% SDS, 0.01% bromophenol blue; pH 8), and analyzed by SDS-PAGE and immunoblotting. Blots were processed according to standard protocols using appropriate dilutions of rabbit anti-Ydj1p (courtesy of Dr. Avrom Caplan) and HRP-conjugated donkey or goat anti-rabbit antibodies (GE Healthcare, Little Chalfont, UK; Kindle Biosciences, Greenwich, CT). Antibody dilutions were prepared using TBST (10 mM Tris, 150 mM NaCl, 0.1% Tween-20; pH 7.5) containing 1% milk (w/v). Immune complexes on blots were detected using X-ray film after treatment with HyGLO development solution (Denville Scientific, South Plainfield, NJ) or using a KwikQuant Imager at multiple exposure times after treatment with the KwikQuant Western Blot Detection Kit (Kindle Biosciences).