title: Identifying Protein-Protein Associations at the Nuclear Envelope with BioID.
abstract: The nuclear envelope (NE) is a critical cellular structure whose constituents and roles in a myriad of cellular processes seem ever expanding. To determine the underlying mechanisms by which the NE constituents participate in various cellular events, it is necessary to understand the nature of their protein-protein associations. BioID (proximity-dependent biotin identification) is a recently established method to generate a history of protein-protein associations as they occur over time in living cells. BioID is based on fusion of a bait protein to a promiscuous biotin ligase. Expression of the BioID fusion protein in a relevant cellular environment enables biotinylation of vicinal and interacting proteins of the bait protein, permitting isolation and identification by conventional biotin-affinity capture and mass-spec analysis. In this way, BioID provides unique capabilities to identify protein-protein associations at the NE. In this chapter we provide a detailed protocol for the application of BioID to the study of NE proteins.
authors.affiliation: Sanford Research, Sanford Children's Health Research Center, 2301 E. 60th Street N., Sioux Falls, SD, 57104, USA.
authors.forename: Dae In
authors.initials: DI
authors.lastname: Kim
authors.affiliation: Sanford Research, Sanford Children's Health Research Center, 2301 E. 60th Street N., Sioux Falls, SD, 57104, USA.
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Jensen
authors.affiliation: Sanford Research, Sanford Children's Health Research Center, 2301 E. 60th Street N., Sioux Falls, SD, 57104, USA. kyle.roux@sanfordhealth.org.|Department of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD, 57105, USA. kyle.roux@sanfordhealth.org.
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Roux
mesh_terms: D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism*; D034881:Nuclear Lamina / Q000378:metabolism; D022022:Nuclear Pore / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins; D015203:Reproducibility of Results
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D001710:Biotin
keywords: BioID; Biotinylation; Nuclear envelope; Nuclear lamina; Nuclear pore complex; Protein-protein interactions