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001-es BibID:	BIBFORM004878
035-os BibID:	(scopus)26444586247 (wos)000232299300007
Első szerző:	Nagy Péter (biofizikus)
Cím:	Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins / Nagy, P., Bene, L., Hyun, W. C., Vereb, G., Braun, M., Antz, C., Paysan, J., Damjanovich, S., Park, J. W., Szollosi, J.
Dátum:	2005
ISSN:	1552-4922
Megjegyzések:	The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk analysis Biophysics Calibration Cells chemistry Cyclin-Dependent Kinase 2 Cyclin-Dependent Kinase Inhibitor p27 Energy Transfer Flow Cytometry Fluorescence Fluorescence Resonance Energy Transfer Hungary metabolism methods Protein Binding Proteins Research Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Support
Megjelenés:	Cytometry. Part A. - 67 : 2 (2005), p. 86-96. -
További szerzők:	Bene László (1963-) (biofizikus) Hyun, William C. Vereb György (1965-) (biofizikus, orvos) Braun, Manuel Antz, Christof Paysan, Jacques Damjanovich Sándor (1936-2017) (biofizikus) Park, John W. Szöllősi János (1953-) (biofizikus)
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001-es BibID:	BIBFORM004634
035-os BibID:	(scopus)0032101894 (wos)000073937700007
Első szerző:	Nagy Péter (biofizikus)
Cím:	EGF-induced redistribution of erbB2 on breast tumor cells : flow and image cytometric energy transfer measurements / Nagy, P., Bene, L., Balazs, M., Hyun, W. C., Lockett, S. J., Chiang, N. Y., Waldman, F., Feuerstein, B. G., Damjanovich, S., Szollosi, J.
Dátum:	1998
Megjegyzések:	erbB2, a member of the epidermal growth factor (EGF) receptor-type tyrosine kinase receptor family, is overexpressed in breast carcinomas with poor prognosis. We examined the cell surface association of this receptor with itself and with other cell surface proteins by the Forster-type fluorescence resonance energy transfer using whole antibodies and Fab fragments. We found that erbB2 molecules homoassociate in unstimulated SK-BR-3, BT474 and BT474-M (a metastatic version of the parent BT474 line) breast tumor cells, and that the interaction was enhanced by EGF treatment in suspensions of SK-BR-3 and BT474-M cells. BT474 cells (with low EGF receptor expression) and attached SK-BR-3 cells do not respond to EGF. Image microscopic energy transfer measurements found considerable pixel-by-pixel heterogeneity in the homoassociation state of erbB2. In accordance with the EGF-induced redistribution of erbB2, EGF receptor was found to be in close proximity to erbB2 in FRET measurements. By labeling different epitopes on erbB2 and the lipid bilayer, we were able to prepare an epitope map of erbB2 molecule. Our data suggest the existence of dynamic cell surface patterns of erbB2 and point to functions fulfilled by these molecular complexes.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk analysis biosynthesis Breast Neoplasms Carcinoma Cell Membrane chemistry drug effects Energy Transfer Epidermal Growth Factor Female Flow Cytometry Fluorescence Human Hungary metabolism methods Models Molecular pharmacology Receptor erbB-2 Receptors Transferrin Support Non-U.S.Gov't Tumor Cells,Cultured Squamous Cell ultrastructure
Megjelenés:	Cytometry. - 32 : 2 (1998), p. 120-131. -
További szerzők:	Bene László (1963-) (biofizikus) Balázs Margit (1952-) (sejtbiológus, molekuláris genetikus) Hyun, William C. Lockett, Steven J. Chiang, Nancy Y. Waldman, Frederick Feuerstein, Burt G. Damjanovich Sándor (1936-2017) (biofizikus) Szöllősi János (1953-) (biofizikus)
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