title: Green fluorescent protein as a quantitative tool.
abstract: Manipulating the expression of a protein can provide a powerful tool for understanding its function, provided that the protein is expressed at physiologically-significant concentrations. We have developed a simple method to measure (1) the concentration of an overexpressed protein in single cells and (2) the covariation of particular physiological properties with a protein's expression. As an example of how this method can be used, teratocarcinoma cells were transfected with the neuron-specific calcium binding protein calretinin (CR) tagged with green fluorescent protein (GFP). By measuring GFP fluorescence in microcapillaries, we created a standard curve for GFP fluorescence that permitted quantification of CR concentrations in individual cells. Fura-2 measurements in the same cells showed a strong positive correlation between CR-GFP fusion protein expression levels and calcium clearance capacity. This method should allow reliable quantitative analysis of GFP fusion protein expression.
authors.affiliation: Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City 84132, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Hack
authors.forename: B
authors.initials: B
authors.lastname: Billups
authors.forename: P B
authors.initials: PB
authors.lastname: Guthrie
authors.forename: J H
authors.initials: JH
authors.lastname: Rogers
authors.forename: E M
authors.initials: EM
authors.lastname: Muir
authors.forename: T N
authors.initials: TN
authors.lastname: Parks
authors.forename: S B
authors.initials: SB
authors.lastname: Kater
mesh_terms: D000818:Animals; D064032:Calbindin 2; D002118:Calcium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008297:Male; D064030:S100 Calcium Binding Protein G / Q000032:analysis / Q000378:metabolism*; D018243:Teratocarcinoma; D013736:Testicular Neoplasms; D014407:Tumor Cells, Cultured
chemical_list: C576764:CALB2 protein, human; D064032:Calbindin 2; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D064030:S100 Calcium Binding Protein G; D049452:Green Fluorescent Proteins; D002118:Calcium