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Diffusion of CaM and CaMK-II

Explore the diffusion mechanisms of CaM and CaMK-II using Fick’s model and fluorescence correlation spectroscopy. Learn how to determine diffusion constants, address procedural concerns like bleaching, and connect with related topics such as FRAP. Acknowledging Dr. Waxham, Hugo Sanabria, Matt Swulius, and Ben Goins for their contributions.

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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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