title: Quantification of Protein Levels in Single Living Cells.
abstract: Accurate measurement of the amount of specific protein a cell produces is important for investigating basic molecular processes. We have developed a technique that allows for quantitation of protein levels in single cells in vivo. This protein quantitation ratioing (PQR) technique uses a genetic tag that produces a stoichiometric ratio of a fluorescent protein reporter and the protein of interest during protein translation. The fluorescence intensity is proportional to the number of molecules produced of the protein of interest and is used to determine the relative amount of protein within the cell. We use PQR to quantify protein expression of different genes using quantitative imaging, electrophysiology, and phenotype. We use genome editing to insert Protein Quantitation Reporters into endogenous genomic loci in three different genomes for quantitation of endogenous protein levels. The PQR technique will allow for a wide range of quantitative experiments examining gene-to-phenotype relationships with greater accuracy.
authors.affiliation: Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre, Montréal, QC H3G 1A4, Canada.
authors.forename: Chiu-An
authors.initials: CA
authors.lastname: Lo
authors.affiliation: Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre, Montréal, QC H3G 1A4, Canada.
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Kays
authors.affiliation: Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre, Montréal, QC H3G 1A4, Canada.
authors.forename: Farida
authors.initials: F
authors.lastname: Emran
authors.affiliation: Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre, Montréal, QC H3G 1A4, Canada.
authors.forename: Tsung-Jung
authors.initials: TJ
authors.lastname: Lin
authors.affiliation: Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre, Montréal, QC H3G 1A4, Canada.
authors.forename: Vedrana
authors.initials: V
authors.lastname: Cvetkovska
authors.affiliation: Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre, Montréal, QC H3G 1A4, Canada; Departments of Medicine and Neurology and Neurosurgery, McGill University, Montréal, QC H3G 1A4, Canada. Electronic address: brian.chen@mcgill.ca.
authors.forename: Brian Edwin
authors.initials: BE
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004330:Drosophila / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D059010:Single-Cell Analysis
chemical_list: D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins