fluoroproteins

Fluoroproteins are proteins that emit fluorescence upon excitation with light. The term encompasses proteins that are inherently fluorescent, typically fluorescent proteins engineered by genetic encodings, as well as proteins that carry covalently attached fluorescent probes. Fluoroproteins are widely used as reporters to visualize biological processes in living cells and organisms.

Intrinsic fluoroproteins include fluorescent proteins such as GFP and its spectral variants (EGFP, YFP, CFP, mCherry, mNeonGreen, and others). These proteins form a chromophore in situ and can be expressed as fusions to target proteins, enabling real-time monitoring of localization, trafficking, and dynamics without exogenous dyes.

Fluorophore-labeled proteins are produced by covalent attachment of synthetic fluorescent dyes to the protein. Common dyes include Alexa Fluor, Cy dyes, FITC, and rhodamines, linked through reactive groups to lysine or cysteine residues. This approach yields bright signals and allows multi-color labeling, but often requires purification to remove free dye and can perturb protein function.

Some researchers also prepare fluorinated or fluorinated-labeled proteins for specialized techniques. Incorporation of fluorine-containing amino acids or tags enables 19F NMR, MRI, or enhanced stability, though these approaches are more specialized and less common for routine imaging.

Applications include fluorescence microscopy, confocal and super-resolution imaging, flow cytometry, western blotting with fluorescent detection, and immunoassays. Fluoroproteins support studies of protein localization, interactions, turnover, and signaling; fusion proteins enable biosensors and reporters for cellular events, transcriptional activity, and in vivo imaging.

Important considerations when working with fluoroproteins include spectral properties and overlap between fluorophores, brightness and photostability, maturation time for fluorescent proteins, potential perturbation of protein function, expression levels, and potential cytotoxicity of dyes.

Fluoroproteins intersect with fields such as fluorescence imaging, proteomics, and biosensor design.