Use of protein biotinylation in vivo for immunoelectron microscopic localization of a specific protein isoform.
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.
Université Paris-Sud 11, CNRS, Interactions Moléculaires et Cancer UMR 8126, Institut de Cancérologie Gustave-Roussy, Villejuif, France.
Antoine
A
Viens
Francis
F
Harper
Evelyne
E
Pichard
Martine
M
Comisso
Gérard
G
Pierron
Vasily
V
Ogryzko
D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D006657:Histones / Q000378:metabolism; D006801:Humans; D016253:Microscopy, Immunoelectron; D009687:Nuclear Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D016610:Tissue Embedding; D014162:Transfection
D002843:Chromatin; D029968:Escherichia coli Proteins; D006657:Histones; D009687:Nuclear Proteins; D009842:Oligopeptides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli