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Use of Protein Biotinylation In Vivo for Immunoelectron Microscopic Localization of a Specific Protein Isoform

Antoine Viens, Francis Harper, Evelyne Pichard, Martine Comisso, Gérard Pierron¹ and Vasily Ogryzko¹

Université Paris-Sud 11, CNRS, Interactions Moléculaires et Cancer UMR 8126, Institut de Cancérologie Gustave-Roussy, Villejuif, France (AV,MC,VO), and Réplication de l'ADN et Ultrastructure du Noyau, CNRS FRE-2937, Institut André Lwoff, Villejuif, France (FH,EP,GP)

Correspondence to: Vasily Ogryzko, 39 Rue Camille Desmoulins, IGR, PR1, Room 325, Villejuif 94805, France. E-mail: vogryzko{at}gmail.com

Tagging of proteins in vivo by covalent attachment of a biotin moiety has emerged as a new prospective tool for protein detection and purification. Previously, we established a strategy for expression of in vivo biotinylated proteins in mammalian cells. It is based on coexpression of the protein of interest fused to a short biotin acceptor peptide and biotin ligase BirA cloned in the same vector. We show here that the in vivo biotinylation can be used for immunogold postembedding labeling in immunoelectron microscopy experiments. We show that immunoelectron microscopy with biotinylated nuclear proteins is compatible with a wide range of postembedding methods, facilitating combination of morphological and localization studies in a single experiment. We also show that the method works in both transient transfection and stable cell line expression protocols and can be used for colocalization studies. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 56:911–919, 2008)

Key Words: biotin • electron microscopy • postembedding • SUMO2 • PML

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