METHOD FOR QUANTITATIVE ANALYSIS OF REAL TIME INTERACTIONS BETWEEN HIF-1 ALPHA AND P300 PROTEINS

摘要

PURPOSE: A real time analysis method of the interaction between HIF-1alpha and p300 protein is provided to quantitatively analyze the interaction between HIF-1alpha and p300 protein in real time at a cellula level using a fluorescence resonance energy transfer method and to screen the interaction in a cell in real time. CONSTITUTION: A method for measuring the interaction between HIF-1alpha and p300 protein comprises the steps of: preparing an HIF-1alpha plasmid containing HIF-1alpha of which N terminal, C terminal, or the both terminals is (are) labeled with a first fluorescent material; preparing a p300 protein plasmid containing p300 protein of which N terminal, C terminal, or both terminals is (are) labeled with a second fluorescent material; injecting the prepared HIF-1alpha plasmid and p300 plasmid into eukaryotes; and measuring the fluorescence generated from the transfected cells and calculating the fluorescence transfer efficiency according to equation (FRET index = I_DA- (a_DI_DA)- (a_AI_DA), FRET efficiency = FRET index/255). One of the first and second fluorescent materials is a fluorescent energy donor and the other is a fluorescent energy acceptor. The fluorescence spectrum of the fluorescent energy donor and the fluorescent energy acceptor is partially or entirely overlapped.