Fluorescence has two distinct stages, excitation stage and emission stage. It is a phenomenon where fluorescent chromophore absorbs a light photon, typically remains in an excited state for a few nanoseconds and then emits a lower energy photon. Both the efficiency of light absorption (ε) and the efficiency of photon emission from an excited fluorophore (Q) determine the intensity of a sample. Changes in either ε and/or Q will lead to changes in sample emission intensity.
As the most sensitive spectroscopic technique available, reproducible signals can be quantified with best fluorescence spectrometer from samples containing a small amount of material (pM–nM range) some fluorophores, much lower than those required for EPR, NMR, CD and other spectral techniques. The intensity, lifetime, energy and rotational freedom can be analyzed to reveal different aspects of a structure, interaction or mechanism. What's more, to determine its kinetics, the signal change in fluorescence spectroscopy be monitored as a function of time since fluorescence is a nondestructive phenomenon. The dynamic nature of the signal, its localized nature, and its redundancy make fluorescence spectroscopy popular for the analysis of protein and peptide. It is one of the most powerful techniques to study protein folding, dynamics, assembly, and interactions, as well as membrane structure.
Fluorescence spectroscopy is widely applicable since almost all proteins have tyrosine and tryptophan, residues natural fluorophores, which allow study of protein conformation changes. Trp is a commonly used intrinsic fluorophores in protein structure study because of the highest quantum yield and its emission maximum sensitive to the polarity of its environment. Trp is usually only used as an intrinsic fluorophores in proteins or peptides lacking Trp, since Tyr has significantly lower quantum yield and usually quenched by Trp residues. The Tyr emission maximum depends on pH and is not sensitive to the polarity of environment. With mutagenesis and chemical modifications, site-specific labeling with external fluorophores is readily achievable. Since the fluorescence emission lifetime is in the nanosecond range, relatively faster than most conformational transitions, it is a powerful method to study fast protein conformational changes.
The most difficult and critical aspect of every fluorescence experiment is the biochemistry, not the spectroscopy. That's because the observed signal is a combination of the individual signals from more than 1011 separate fluorophores, except in a true single‐molecule experiment. Therefore, biochemical and chemical analysis of the homogeneity in the sample is important for proper interpretation of the signal and observed changes. With years' experience in fluorescence spectroscopy, Creative Proteomics is good at interpretation of the signal and observed changes. Besides, Creative Proteomics usually does biochemical and chemical controls and analysis to ensure that the spectral data are interpreted correctly.
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