Fluorescent substance and fluorescent labeling technology
The compounds upon which fluorescent labeling depends are called fluorescent substances. Fluorescent substances refer to compounds with a conjugated double bond system chemical structure, which can be excited into an excited state when irradiated by ultraviolet light or blue-violet light, and emit fluorescence when returning to the ground state from the excited state.
Fluorescent labeling technology refers to the use of fluorescent substances to covalently bind or physically adsorb on a certain group of the molecule to be studied, and use its fluorescent properties to provide information about the researched object. Fluorescent markers have the advantages of no radiation pollution and easy operation, which makes the application of fluorescent markers more and more extensive in many research fields. People use fluorescently labeled polypeptides to detect the activity of target proteins, and the high-throughput activity screening method developed by them is applied to drug screening and drug development of target proteins for disease treatment (for example, various kinases, phosphatases, peptidases, etc.).
What is Fluorescence resonance energy transfer (FRET)?
Fluorescence resonance energy transfer (FRET) is a non-radiative energy transition that transfers the energy from the excited state of the donor to the excited state of the acceptor through the electric dipole interaction between molecules. This process does not involve photons, so it is non-radiative. The analytical method has the advantages of rapidity, sensitivity and simplicity.
Fluorescence resonance energy transfer (FRET) is a technology developed earlier. With the development of green fluorescent protein application technology, FRET has become a tool for detecting nanoscale distances and nanoscale distance changes of biological macromolecules in vivo. It is a powerful tool and has a wide range of applications in biomacromolecule interaction analysis, cell physiology research, and immune analysis.
Principle of FRET
Fluorescence resonance energy transfer is an energy transfer phenomenon between two fluorescent molecules that are very close to each other. When the emission spectrum of the donor fluorescent molecule overlaps with the absorption spectrum of the acceptor fluorescent molecule, and the distance between the two molecules is within 10nm, a non-radioactive energy transfer occurs, that is, the FRET phenomenon, so that the fluorescence intensity of the donor is much lower than when it exists alone (fluorescence quenching), while the fluorescence emitted by the acceptor is greatly enhanced (sensitized fluorescence).
Application of FRET
With the continuous deepening of life science research, the mechanism of various life phenomena, especially the study of protein-protein interaction in cells has become particularly important. But in order to make major breakthroughs in these areas of research, technological progress is essential.
Some traditional research methods continue to develop, providing extremely favorable conditions for the study of protein-protein interactions, but at the same time, these research methods also have many defects: such as yeast two-hybrid, phosphorylated antibody, immunofluorescence, radioactive labeling, etc. The premise of the application of the method is to break the cell or cause damage to the cell, and it is impossible to conduct a dynamic study of the protein-protein interaction in the cell in real time under the physiological conditions of living cells. The application of FRET technology combined with genetic engineering and other technologies just makes up for this defect.
Commonly used standard dye combinations for FRET