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Amphipathic Fluorophores for Probing Cell Membrane Properties

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

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  1. Amphipathic Fluorophores for Probing Cell Membrane Properties Demetrius Cummings University of California, Riverside

  2. Outline… • Background • Experimental process • Results and future goals

  3. Background… • Prestin • Enhances electromotility • Sound reception • Other properties? http://med.stanford.edu/ohns/research/

  4. Fluorophores… • Fluorescent component; multiple types • Often used in diagnostics: tissues, macromolecules, diseases http://exobio.ucsd.edu/MOD/detection.htm

  5. Plasma Membrane…the“Fluid Mosaic” (Dowhan & Bogdanov, 2005) • Amphipathic structure

  6. 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.

  7. 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

  8. Dye Synthesis…

  9. Nucleophilic Aromatic Substitution… • Halide-dependent • Yields… • --chloro- = 37% • --bromo- = 97%

  10. The Process…(part 2) Dye preparation Utilize HEK cells Analysis: photophysical properties Analysis: photophysical properties Combine dye and cells Analysis: photophysical properties

  11. Results… Water (more polar solvent) V.S. Hexane (less polar solvent)

  12. The Process…(part 3) Dye preparation Utilize HEK cells Analysis: photophysical properties Analysis: photophysical properties Combine dye and cells Analysis: photophysical properties

  13. Low absorbance • High incidence of light scattering

  14. The Process…(part 4) Dye preparation Utilize HEK cells Analysis: photophysical properties Analysis: photophysical properties Combine dye and cells Analysis: photophysical properties

  15. Results… • Blue shift evident in HEK cells with dye • Location: less polar environment

  16. 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.

  17. Future Research… • Continue fluorescence analysis of HEK cells: excitation, emission, anisotropy • Effect of dye on cells? • Apply same procedure to cells transfected with prestin

  18. Acknowledgements… • Dr. Bahman Anvari • Dr. Valentine Vullev • Dr. Feng Qian • Dr. Valentine Vullev’s lab • Program coordinators

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