Work

Protocol to differentially quantify spatially resolved viral protein-cellular protein interactions via proximity ligation assays

Year: 2026

Type: article

Abstract: The spatial organization of viral and cellular proteins shapes signal transduction. Here, we present a protocol to quantify differential protein-protein interactions by measuring their spatial association using proximity ligation assays (PLAs). We describe steps for seeding, transfection, proximity labeling, and confocal microscopy imaging and provide procedures for quantitative analysis. Our approach complements co-immunoprecipitation-based interactome data by enabling in situ quantification of differential binding between cellular and viral interaction partners. For complete details on the use and execution of this protocol, please refer to Klute et al. 1 • Assess the frequency of close proximity events between viral and cellular proteins • Quantify relative differences in interactions of protein variants in situ • Detailed suggestions and macros for (semi-)automated PLA quantification • Rapid, flexible system suitable for analyzing virtually any protein-protein interaction Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The spatial organization of viral and cellular proteins shapes signal transduction. Here, we present a protocol to quantify differential protein-protein interactions by measuring their spatial association using proximity ligation assays (PLAs). We describe steps for seeding, transfection, proximity labeling, and confocal microscopy imaging and provide procedures for quantitative analysis. Our approach complements co-immunoprecipitation-based interactome data by enabling in situ quantification of differential binding between cellular and viral interaction partners.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2026.104361

Authors: Helene Hoenigsperger, Susanne Klute, Zoé Engels, Meta Volcic, Konstantin Maria Johannes Sparrer

Institutions: Universität Ulm, German Center for Neurodegenerative Diseases

Language: English

Topic: Biotin and Related Studies

Subfield: Cell Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,550