Proteins fluorescence detection

A simple alternative to direct detection of intrinsic protein fluorescence detection is the technique of indirect fluorescence detection proposed by Kuhr and Yeung.20 In this approach, the analysis buffer contains a fluorescent anion that produces a high background fluorescence signal. Nonfluorescent analyte anions displace the fluorescent species, producing a zone of reduced signal. Sensitivity in indirect fluorescence detection is determined by the dynamic reserve (ratio of signal... 

Luminescence measurements on proteins occupy a large part of the biochemical literature. In what surely was one of the earliest scientific reports of protein photoluminescence uncomplicated by concurrent insect or microorganism luminescence, Beccari (64), in 1746, detected a visible blue phosphorescence from chilled hands when they were brought into a dark room after exposure to sunlight. Stokes (10) remarked that the dark (ultraviolet) portion of the solar spectrum was most efficient in generating fluorescent emission and identified fluorescence from animal matter in 1852. In general, intrinsic protein fluorescence predominantly occurs between 300 nm and 400 nm and is very difficult to detect visually. The first... 

Lee, I.H., Pinto, D., Arriaga, E.A., Zhang. Z., Dovichi, NJ. (1998). Picomolar analysis of proteins using electrophoretically mediated microanalysis and capillary electrophoresis with laser-induced fluorescence detection. Anal. Chem. 70, 4546 4548. 

Sluszny, C., Yeung, E.S. (2004). One-and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection. Anal. Chem. 76, 1359-1365. 

Mao, Y., Zhang, X.M. (2003). Comprehensive two-dimensional separation system hy coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection. Electrophoresis 24, 3289-3295. 

Meadows F, Narayanan N, Patonay G (2000) Determination of protein-dye association by near infrared fluorescence-detected circular dichroism. Talanta 50 1149-1155... 

Yan W, Sloat AL, Yagi S, Nakazumi H, Colyer CL (2006) Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis 27 1347-1354... 

B. Schmidt and D. Riesner, A fluorescence detection system for the analytical ultracentrifuge and its application to proteins, nucleic acids, viroids and viruses (in Ref. [77]). 

Kato, N., Pontier, D. and Lam, E. (2002). Spectral profiling for the simultaneous observation of four distinct fluorescent proteins and detection of protein-protein interaction via fluorescence resonance energy transfer in tobacco leaf nuclei. Plant Physiol. 129, 931-42. 

D.I. Sanchez Machado, B. Chavira Willys, J. Lopez Cervantes, High Performance Liquid Chromatography with Fluorescence Detection for Quantitation of Tryptophan and Tyrosine in a Shrimp Waste Protein Concentrate, Journal of Chromatography, B, 863(1), 88 93 (2008). 

The nonreactive base structures of cyanine dyes (or carbocyanines) have been used for many years as components in photographic emulsions to increase the range and sensitivity of film and also in CD-R and DVD-R optical disks to record digital information. A major innovation came when Ernst et al. (1989) and Waggoner et al. (1993) recognized that cyanine dyes would make excellent labels for fluorescence detection, and for this reason, they synthesized reactive dye derivatives, which then could be covalently attached to proteins and other molecules. 

DeMar Jr. J.C., Disher, R.M., and Wensel, T.G. (1992) HPLC analysis of protein-linked fatty acids using fluorescence detection of 4-(diazomethyl)-7-diethylaminocoumarin derivatives Abstract 465. Biophys. J. 61(A81). 

Keywords Biomembrane DNA Fluorescent detection J-aggregate Protein... 

The polymers used for fluorescence detection discussed thus far have all used either a synthetically linked polymer as the signal transducer for enhanced detection or some type of hybridization of the polymer with a receptor for ligand-receptor binding signaling. One of the most notable aspects of CPs is their ability to act as direct reporters for the detection of small molecules, or for conformational changes and protein aggregates. 

A.D. Presley, K.M. Fuller and E. A. Arriaga, MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection. J. Chromatogr.B, 793 (2003) 141-150. 

J. R. Lakowicz and G. Weber, Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale, Biochemistry 12, 4171-4179 (1973). 

Proteins that do not contain tryptophan or tyrosine must be derivatized prior to fluorescence detection. A common derivatization chemistry involves the reaction... 

The characteristics that discourage the use of RPLC for preparative isolation of bioactive proteins favor its use as an analytical tool for studying protein conformation. Chromatographic profiles can provide information on conformational stability of a protein and the kinetics of folding and unfolding processes. Information about solvent exposure of certain amino acid residues (e.g., tryptophan) as a function of the folding state can be obtained by on-line spectral analysis using diode array UV-vis detection or fluorescence detection. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

The high sensitivity and selectivity of fluorescence detection make this the obvious choice for improving detection of proteins. Three approaches have been used direct detection of intrinsic protein fluorescence, indirect fluorescence detection, and protein derivatization for fluorescence detection. 

Fluorescence detection offers the possibility of high sensitivity and, in the case of complex samples, improved selectivity. However, this mode of detection requires that the analyte exhibit native fluorescence or contain a group to which a fluorophore can be attached by chemical derivatization. Because only tryptophan and tyrosine exhibit significant native fluorescence, fluorescence detection of proteins usually requires derivatization. 

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