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Analysis of Proteins Interactions by Flow Cytometry with FRET (CAT#: STEM-CBT-0065-WXH)

Introduction

Protein-protein interactions (PPIs) handle a wide range of biological processes, including cell-to-cell interactions and metabolic and developmental control. Protein-protein interaction is becoming one of the major objectives of system biology. <br />Fluorescence resonance energy transfer (FRET) techniques have been widely used in recent years to detection of macromolecular interactions in living organisms, protease activity detection, cellular physiology cell physiology, immunoassay, etc. It is a non-invasive detection technique. Combined with multiparametric flow cytometry, FRET offers an attractive tool to study protein interactions in real-time in a large number of individual cells and in a short period of time. Flow cytometric FRET also offers cell sorting of single cell or population of live cells that show FRET for further study. <br />Flow cytometry with FRET has several significant advantages compared to existing methods. It allows to detect protein interactions in all cellular compartments, it is fast and quantitative, non-invasive and highly reproducible.




Principle

Fluorescence resonance energy transfer (FRET) between fluorescent proteins is an elegant non-invasive technique available to detect direct protein-protein interactions in living cells. It is based upon the energy transfer from an excited donor fluorophore to an adjacent acceptor fluorophore, resulting in decreased fluorescence emission by the donor and enhanced fluorescence emission by the acceptor. For direct protein-protein interactions, FRET is highly dependent on the distance between the two fluorophores. Accordingly, this phenomenon only occurs when the distance between donor and acceptor ranges between 1-10 nm and the emission spectra of the donor overlaps with the excitation spectra of the acceptor. In addition, FRET is also dependent on the geometry of the fluorophores in a donor-acceptor FRET pair.

Applications

• Predicting the protein function of target protein.
• Predicting the drug ability of molecules.
• Infer the protein function within the cell.
• Study of the molecular mechanisms of cellular processes.
• Figure out the biochemistry of the cell.

Procedure

1.Plasmid constructs
2.Cell cuture and transfection
3.FRET signal measurement by flow cytometry

Materials

FRET fluorophores: Cyan and yellow fluorescent proteins (CFPs and YFPs)

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