Enhanced fluorescent imaging of proteins in live yeast cells using fluorescently labeled scFv

Fluorescent labeling of proteins is a widespread approach for the microscopic examination of protein function, expression, and localization in the cell. Here, we present a protocol for the labeling of hemagglutinin (HA)-tagged protein of interest (POI) with the single-chain antibody (scFv) 2E2 fused to different fluorescent proteins (FPs) in Saccharomyces cerevisiae. We describe steps for expressing 2E2-FP, and HA tagging and labeling of POI. We detail in vivo fluorescent imaging of proteins at different cellular compartments and with diverse expression levels. For complete details on the use and execution of this protocol, please refer to Tsirkas et al. (2022).1 [Display omitted] •Enhanced fluorescent labeling of HA-tagged proteins in live yeast cells with 2E2-FP•Collection of plasmids containing 2E2 scFv fused with green, red, and blue FPs and HALOtag•Step-to-step guide for HA-labeling of POI and 2E2-FP expression•Identification and quantification of ring-shaped structures and foci/puncta formation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Fluorescent labeling of proteins is a widespread approach for the microscopic examination of protein function, expression, and localization in the cell. Here, we present a protocol for the labeling of hemagglutinin (HA)-tagged protein of interest (POI) with the single-chain antibody (scFv) 2E2 fused to different fluorescent proteins (FPs) in Saccharomyces cerevisiae. We describe steps for expressing 2E2-FP, and HA tagging and labeling of POI. We detail in vivo fluorescent imaging of proteins at different cellular compartments and with diverse expression levels.