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Amphipathic Fluorophores for Probing Cell Membrane Properties. Demetrius Cummings University of California, Riverside. Outline…. Background Experimental process Results and future goals. Background…. Prestin Enhances electromotility Sound reception Other properties?.
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Amphipathic Fluorophores for Probing Cell Membrane Properties Demetrius Cummings University of California, Riverside
Outline… • Background • Experimental process • Results and future goals
Background… • Prestin • Enhances electromotility • Sound reception • Other properties? http://med.stanford.edu/ohns/research/
Fluorophores… • Fluorescent component; multiple types • Often used in diagnostics: tissues, macromolecules, diseases http://exobio.ucsd.edu/MOD/detection.htm
Plasma Membrane…the“Fluid Mosaic” (Dowhan & Bogdanov, 2005) • Amphipathic structure
The Goals… • Synthesize an amphipathic dye for the purpose of binding to the plasma membrane. • Monitor the photophysical properties of the dye and HEK cells in various conditions.
The Process…(part 1) Dye preparation Dye preparation Utilize HEK cells Utilize HEK cells Analysis: photophysical properties Analysis: photophysical properties Combine dye and cells Analysis: photophysical properties
Nucleophilic Aromatic Substitution… • Halide-dependent • Yields… • --chloro- = 37% • --bromo- = 97%
The Process…(part 2) Dye preparation Utilize HEK cells Analysis: photophysical properties Analysis: photophysical properties Combine dye and cells Analysis: photophysical properties
Results… Water (more polar solvent) V.S. Hexane (less polar solvent)
The Process…(part 3) Dye preparation Utilize HEK cells Analysis: photophysical properties Analysis: photophysical properties Combine dye and cells Analysis: photophysical properties
Low absorbance • High incidence of light scattering
The Process…(part 4) Dye preparation Utilize HEK cells Analysis: photophysical properties Analysis: photophysical properties Combine dye and cells Analysis: photophysical properties
Results… • Blue shift evident in HEK cells with dye • Location: less polar environment
Summary… • The efficiency and mechanism of the dye are halide-dependent. • The absorbance varies directly with dye-solvent and cell-solvent concentration; the specific wavelengths of absorbance remains constant. • Blue shift of the dye-HEK cell mix indicates binding to the less polar environment.
Future Research… • Continue fluorescence analysis of HEK cells: excitation, emission, anisotropy • Effect of dye on cells? • Apply same procedure to cells transfected with prestin
Acknowledgements… • Dr. Bahman Anvari • Dr. Valentine Vullev • Dr. Feng Qian • Dr. Valentine Vullev’s lab • Program coordinators