Epitope tags are widely used for detecting,modifying,or purifying proteins of interest,but their range of application is often limited.Recently,the rationally designed ALFA tag and its ALFA nanobody have expanded the ...Epitope tags are widely used for detecting,modifying,or purifying proteins of interest,but their range of application is often limited.Recently,the rationally designed ALFA tag and its ALFA nanobody have expanded the repertoire of epitope tags and emerged as a highly versatile system characterized in various animal models,outperforming existing tags.Here,we evaluated the ALFA tag/ALFA nanobody technology in plants and demonstrated its application for in planta protein detection across multiple compartments and cellular structures,protein-protein interaction studies,protein immunoprecipitation,inducedproximity approaches,and super-resolution microscopy.Most importantly,we highlight the potential of the ALFA tagging technology for proteins that are difficult to tag due to topological or functional constraints.We provide proof of concept for the ALFA tag technology in the detection and functional analysis of the Arabidopsis IRT1 Fe transporter.Overall,this versatile and validated toolbox of ALFA tag and ALFA nanobody applications will serve as a valuable resource for functional studies in plants.展开更多
基金supported by research grants from the French National Research Agency(grant nos.ANR-21-CE20-0046 to G.V.and 22-PESV0002 to J.N.)the French Laboratory of Excellence(project“TULIP”grant nos.ANR-10-LABX-41 and ANR-11-IDEX-0002-02 to G.V.).
文摘Epitope tags are widely used for detecting,modifying,or purifying proteins of interest,but their range of application is often limited.Recently,the rationally designed ALFA tag and its ALFA nanobody have expanded the repertoire of epitope tags and emerged as a highly versatile system characterized in various animal models,outperforming existing tags.Here,we evaluated the ALFA tag/ALFA nanobody technology in plants and demonstrated its application for in planta protein detection across multiple compartments and cellular structures,protein-protein interaction studies,protein immunoprecipitation,inducedproximity approaches,and super-resolution microscopy.Most importantly,we highlight the potential of the ALFA tagging technology for proteins that are difficult to tag due to topological or functional constraints.We provide proof of concept for the ALFA tag technology in the detection and functional analysis of the Arabidopsis IRT1 Fe transporter.Overall,this versatile and validated toolbox of ALFA tag and ALFA nanobody applications will serve as a valuable resource for functional studies in plants.
