Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.
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Transcript of Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.
![Page 1: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/1.jpg)
Diffusion of CaM and CaMK-II
Andrew HarrellDr. Waxham Lab
University of Texas Medical School
![Page 2: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/2.jpg)
Fick’s Diffusion Model
J
nVolume V
Units for D =
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![Page 4: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/4.jpg)
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.
![Page 5: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/5.jpg)
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.
![Page 6: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/6.jpg)
FCS Apparatus
• Laser light (λ = 780 nm) chosen to maximize dye activity.
![Page 7: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/7.jpg)
![Page 8: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/8.jpg)
Data Collection
• Measure the fluorescent intensity as a function of time.
• Computer calculates the correlation function vs. (a time delay).
![Page 9: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/9.jpg)
Correlation Curves
• Wavelength 780 nm chosen to maximize activity of the Alexa-488 dye.
• D(CaM) = 75.00• D(CaMKII+CaM) =
15.78• ( )
10-6
10-4
10-2
100
-1
0
1
10-6
10-4
10-2
100
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.1
1.11
(sec)
g( )
Nac 10.0286K 2.95taud 0.00015778
epsilon 2.3011
Adjusted R2 -102.1231
Vtrue (fL) 0.057516C (M) 1.024e-007Ntrue 3.5456
D (um2/sec) 73.071
take37-take36
data
fit
![Page 10: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/10.jpg)
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:
![Page 11: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/11.jpg)
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
![Page 12: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/12.jpg)
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
![Page 13: Diffusion of CaM and CaMK-II Andrew Harrell Dr. Waxham Lab University of Texas Medical School.](https://reader036.fdocuments.in/reader036/viewer/2022070415/5697bfee1a28abf838cb98b4/html5/thumbnails/13.jpg)
Acknowledgements
• Dr. Waxham – lab director• Hugo Sanabria – supervisor• Matt Swulius – provided images• Ben Goins – thesis material
Questions???