Protocol to determine the subcellular localization of protein interactions in murine keratinocytes

• Published in: STAR protocols Vol. 4; no. 2; p. 102309
• Main Author: Müller, Lisa
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.06.2023; Elsevier
• Subjects: Cell Biology; Cell separation/fractionation; Molecular Biology; Protocol; Molecular Biology; Cell separation/fractionation; Cell Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102309

Protein activities and interactions are determined by their subcellular localization. Elucidating the network of protein-protein interactions at a spatial resolution is essential for understanding the complexity of protein functions, their regulation, and cellular processes. Here, we present a protocol to determine the subcellular localization of protein interactions in non-transformed murine keratinocytes. We describe steps for nucleus/cytoplasm fractionation, immunoprecipitation from these fractions, and immunoblotting. We then detail binding quantification. For complete details on the use and execution of this protocol, please refer to Müller et al. (2023).1 [Display omitted] •Quick assay to identify subcellular localization of protein-protein interactions•Step-by-step guide for high-quality and reproducible sample preparation•Robust protocol to fractionate, purify, immunoblot, and quantify proteins Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Protein activities and interactions are determined by their subcellular localization. Elucidating the network of protein-protein interactions at a spatial resolution is essential for understanding the complexity of protein functions, their regulation, and cellular processes. Here, we present a protocol to determine the subcellular localization of protein interactions in non-transformed murine keratinocytes. We describe steps for nucleus/cytoplasm fractionation, immunoprecipitation from these fractions, and immunoblotting. We then detail binding quantification.