Fluorescent protein detection

Karpova, T. S., Baumann, C. T., He, L., Wu, X., Grammer, A., Lipsky, P., Hager, G. L. and McNally, J. G. (2003). Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. J. Microsc. 209, 56-70. 

Turner, E.H., Lauterbach, K., Pugsley, H., Palmer, V.R. and Dovichi, N.J. Ultrasensitive green fluorescent protein detection in a single bacterium by capfllary electrophoresis with laser-induced fluorescence. Ana/. Chem. 2007 79 778-781. 

From the results obtained, it was found that compound 10a showed very high fluorescence intensity in the presence of the BSA and BSA/SDS mixture ( F 0.27) together with a noticeable emission enhancement. The presence of dimethyl indo-lenyl increased the affinity of the dyes to both native and denatured proteins. The authors proposed compound 10a for further studies as fluorescent probes for protein detection. 

Oswald B, Patsenker L, Duschl J, Szmacinski H, Wolfbeis OS, Terpetschnig E (1999) Synthesis, spectral properties, and detection limits of reactive squaraine dyes, a new class of diode laser compatible fluorescent protein labels. Bioconjugate Chem 10 925-931... 

Yang, T. T., Sinai, P., Green, G., Kitts, P. A., Chen, Y. T., Lybarger, L., Chervenak, R., Patterson, G. H., Piston, D. W. and Kain, S. R. (1998). Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein. J. Biol. Chem. 273, 8212-6. 

Goedhart, J., Vermeer, J. E., Adjobo-Hermans, M. J., van Weeren, L. and Gadella, T. W., Jr. (2007). Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples. PLoS ONE 2, elOll. 

Romoser, V. A., Hinkle, P. M. and Persechini, A. (1997). Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J. Biol. Chem. 272, 13270-4. 

Rizzo, M. A., Springer, G., Segawa, K., Zipfel, W. R. and Piston, D. W. (2006). Optimization of pairings and detection conditions for measurment of FRET between cyan and yellow fluorescent proteins. Microsc. Micro-anal. 12, 238-54. 

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. 

Pepperkok, R., Squire, A., Geley, S. and Bastiaens, P. I. (1999). Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Curr. Biol. 9, 269-72. 

BRET [31, 32]), lock-in detection techniques exploiting optical switches [33], and schemes for alternating D/A excitation (ALEX [34]). The increased attention to quantitative FRET imaging encompasses the use of polarization [35-39], the perennial issue of calibration and standards [40-44], and practical guides to operational principles and protocols ([45, 46] and other references above). The fundamental distinctions between the requirements for live and fixed cell imaging cannot be overemphasized, as is exemplified in a report of erroneous FRET determinations with visible fluorescent proteins (VFPs) in fixed cells [47],... 

The bilin content of these fluorescent proteins ranges from a low of 4 prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin derivatives, therefore, can be used to create the most intensely fluorescent probes possible using these proteins. (Strept)avidin-phycoerythrin conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound to receptor proteins per cell (Zola et al., 1990). 

The fast, sensitive, reliable, and reproducible detection of (bio)molecules including quantification as well as biomolecule localization, the measurement of their interplay with one another or with other species, and the assessment of biomolecule function in bioassays as well as in vitro and in vivo plays an ever increasing role in the life sciences. The vast majority of applications exploit extrinsic fluorophores like organic dyes, fluorescent proteins, and also increasingly QDs, as the number of bright intrinsic fluorophores emitting in the visible and NIR is limited. In the near future, the use of fluorophore-doped nanoparticles is also expected to constantly increase, with their applicability in vivo being closely linked to the intensively discussed issue of size-related nanotoxicity [88]. 

In addition to protein detection using specific antibody/antigen and aptamer/protein interactions, array detection was demonstrated based on nonspecific interactions between the CPE/dye-labeled ssDNA complexes and proteins [103]. The design concept is motivated by the fact that external agents can effectively perturb the electron coupling of optical units within the complex of CPE/ssDNA-C and in turn vary the FRET-induced fluorescence of both CPE (donor) and C (acceptor). Owing to discrepancies in local hydrophobic and charged domains of different... 

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