Visualizing Hsp104-BFP Foci in Yeast

A blue fluorescence protein (BFP) cassette was amplified from the pFA6a-link-yomTagBFP2-Kan plasmid and integrated into the genome to generate an Hsp104-BFP fusion protein in all tested strains [77 (link)]. Log-phase cells growing in YPD at 25°C were transferred to a 37°C water bath and cells were collected at 0, 1, 2, and 3 hours after the temperature shift. The samples were resuspended in 200 μL PBS and transferred to a Glass Bottom ViewPlate-96F (PerkinElmer, Waltham, MA) that was precoated with concanavalin A (cat. no. C2010, Sigma-Aldrich). Images of Hsp104-BFP foci were acquired using an ImageXpress Micro XL system (Molecular Devices, San Jose, CA) and analyzed using a custom-built module under MetaXpress High-Content Image Acquisition and Analysis Software (Molecular Devices).