Green fluorescent protein as a quantitative tool.
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.
Hack
N J
NJ
Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City 84132, USA.
Billups
B
B
Guthrie
P B
PB
Rogers
J H
JH
Muir
E M
EM
Parks
T N
TN
Kater
S B
SB
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
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