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Diffusion of CaM and CaMK-II

Diffusion of CaM and CaMK-II. Andrew Harrell Dr. Waxham Lab University of Texas Medical School. Fick’s Diffusion Model. J. Units for D =. Volume V. n. Fluorescence. Excitation of a molecule to a higher energy state by photon energy.

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Diffusion of CaM and CaMK-II

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  1. Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School

  2. Fick’s Diffusion Model J Units for D = Volume V n

  3. Fluorescence • Excitation of a molecule to a higher energy state by photon energy. • Subsequent lowering of energy state, accompanied by an emission of radiation. • Ultraviolet -> Visible light.

  4. Fluorescent Correlation Spectroscopy • Uses multi-photon laser excitation to induce fluorescence. • Fluorescent intensity is recorded as a function of time. • A correlation curve is created, which relates fluorescence at a particular time to fluorescence at other times.

  5. FCS Apparatus • Laser light (λ = 780 nm) chosen to maximize dye activity.

  6. Data Collection • Measure the fluorescent intensity as a function of time. • Computer calculates the correlation function vs. (a time delay).

  7. Correlation Curves • Wavelength 780 nm chosen to maximize activity of the Alexa-488 dye. • D(CaM) = 75.00 • D(CaMKII+CaM) = 15.78 • ( )

  8. Determining Diffusion Constants • Interpolate along the curve to find G(0). • G(0) is inversely proportional to the concentration. • Determine the x-coordinate of the point on the best-fit curve whose corresponds to half of G(0). • The time is called . • Based on a Gaussian approximation to the excitation volume, and the two-photon excitation method, we know that:

  9. Procedural Concerns • Bleaching • Possibility that molecules will be chemically altered by the light, in a way which prevents future fluorescence. • Two-photon excitation helps to avoid bleaching. • Determining the “size” of the activity volume • 3-D Gaussian approximation vs. solution to Maxwell’s equations

  10. Related Topics • Fluorescence Recovery After Photobleaching (FRAP) method. • “Opposite” of FCS; uses an intense pulse to photobleach all of the molecules in a certain volume and then observes fluorescent molecules as they diffuse back into the region. • Measuring simultaneous fluorescence of multiple molecules

  11. Acknowledgements • Dr. Waxham – lab director • Hugo Sanabria – supervisor • Matt Swulius – provided images • Ben Goins – thesis material Questions???

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