title: Fluorescent protein specific Nanotraps to study protein-protein interactions and histone-tail peptide binding.
abstract: Fluorescent proteins are widely used to study protein localization and protein dynamics in living cells. Additional information on peptide binding, DNA binding, enzymatic activity, and complex formation can be obtained with various methods including chromatin immunoprecipitation (ChIP) and affinity purification. Here we describe two specific GFP- and RFP binding proteins based on antibody fragments derived from llama single domain antibodies. The binding proteins can be produced in bacteria and coupled to monovalent matrixes generating so-called Nanotraps. Both Nanotraps allow a fast and efficient (one-step) isolation of fluorescent fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Here we provide protocols for precipitation of fluorescent fusion proteins from crude cell extracts to identify and map protein-protein interactions as well as specific histone tail peptide binding in an easy and reliable manner.
authors.affiliation: CIPS, Center for Integrated Protein Science at the Department of Biology II, Ludwig Maximilians University Munich, Planegg-Martinsried, Germany.
authors.forename: Garwin
authors.initials: G
authors.lastname: Pichler
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
mesh_terms: D000818:Animals; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
chemical_list: D006657:Histones; D008164:Luminescent Proteins; D010455:Peptides; D061905:Single-Domain Antibodies