Green fluorescent protein as a quantitative tool

Manipulating the expression of a protein can provide a powerful tool for un derstanding its function, provided that the protein is expressed at physiol ogically-significant concentrations. We have developed a simple method to m easure (1) the concentration of an overexpressed protein in single cells an d (2) the covariation of particular physiological properties with a protein 's expression. As an example of how this method can be used, teratocarcinom a cells were transfected with the neuron-specific calcium binding protein c alretinin (CR) tagged with green fluorescent protein (GFP). By measuring GF P fluorescence in microcapillaries, we created a standard curve for GFP flu orescence that permitted quantification of CR concentrations in individual cells. Fura-2 measurements in the same cells showed a strong positive corre lation between CR-GFP fusion protein expression levels and calcium clearanc e capacity. This method should allow reliable quantitative analysis of GFP fusion protein expression. (C) 2000 Elsevier Science B.V. All rights reserv ed.