Green fluorescent protein as a quantitative tool

被引:24
作者
Hack, NJ
Billups, B
Guthrie, PB
Rogers, JH
Muir, EM
Parks, TN
Kater, SB
机构
[1] Univ Utah, Sch Med, Dept Neurobiol & Anat, Salt Lake City, UT 84132 USA
[2] Univ Cambridge, Dept Physiol, Cambridge CB2 3EG, England
关键词
GFP; calretinin; calcium homeostasis; fluorescence microscopy;
D O I
10.1016/S0165-0270(99)00178-8
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2000 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:177 / 184
页数:8
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