Continuous observation of heat shock protein 70 expression in its kinetics study

Abstract

The kinetics study of heat shock protein 70 (HSP70) expression is essential to any clinical application of HSP70. HSP70 expression was visualized continuously in cultured bovine aortic endothelial cells (BAECs) on a culture dish adaptable heating stage with green fluorescent protein (GFP) as a reporter. BAECs were transfected with a DNA vector, HSP/sub p/-HSP70-GFP, which has HSP70 promoter followed by HSP70 gene and GFP gene. Cloning was used to construct this vector. Western Blot analysis of transfected and heated cells showed the fusion protein expression. Transfected cells were heated on the stage at 42/spl deg/C and post-heated at 37/spl deg/C on the same stage for varied time periods. The expression of the fusion protein, HSP70-GFP, and its localization were visualized by GFP with a fluorescence microscope. HSP70 expression kinetics can be measured by quantitative analysis of serial fluorescence images captured over different heating and post-heating times. This is a much more efficient method to study HSP70 expression kinetics than the discontinuous steps of cell heating, cell lysis and Western Blot analysis. The kinetics data from continuous observation of HSP70 expression will be compared with our endogenous HSP70 expression kinetics study.