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Use of Dynamic Light Scattering to Detect the Growth of Amyloid Fibrils in HEWL.

Use of Dynamic Light Scattering to Detect the Growth of Amyloid Fibrils in HEWL. . What are they? Why should we study them?. Proteins bonded in a very specific manner How they are formed is not understood Associated with many diseases, including: Alzheimer’s Disease Mad Cow Disease.

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Use of Dynamic Light Scattering to Detect the Growth of Amyloid Fibrils in HEWL.

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  1. Use of Dynamic Light Scattering to Detect the Growth of Amyloid Fibrils in HEWL.

  2. What are they? Why should we study them? • Proteins bonded in a very specific manner • How they are formed is not understood • Associated with many diseases, including: • Alzheimer’s Disease • Mad Cow Disease

  3. Hen Egg White Lysozyme • In water, HEWL is in folded state • Hydrogen bonding • TFE partially unfolds, then stabilizes HEWL • Newly exposed portions of HEWL should bond to each other, forming strands

  4. How Could We Study Them? • We wanted to characterize their rate of formation • Disturbing the proteins would make any data collected questionable • How could we solve this problem?

  5. Dynamic Light Scattering!

  6. What is DLS? • Detects the changing interference pattern of laser light scattered by small particles in solution. • From the rate of change, we can measure the diffusion rate and size of the particles.

  7. Light Scattering in Action!

  8. What is the Autocorrelation Function? • Put simply: the average of the time varying portion of the intensity at some initial time, t, with the time varying portion of the intensity at some later time, t+∆t.

  9. OK, It isn’t really that simple… • This calculation is done for many values of ∆t. • It is repeated many times (in our case, ~400), each time averaging the new result with the average of all the previous results.

  10. Why would you do that? • For small values of ∆t, the signal is still correlated with the signal at t = 0; positive average intensity. • For large values of ∆t, this is not true, and the autocorrelation function will eventually average out to zero.

  11. Key Point • By measuring how long it takes the function to go to zero, we can tell how fast the particles are moving!

  12. For All You Visual Learners…

  13. Microsphere Autocorrelation Function, τ = 3.89 ms.

  14. Single Exponential Autocorrelation Function

  15. Multiple Exponential Autocorrelation Function

  16. Multiple exponential fit

  17. Evolution of the Autocorrelation Function

  18. Time Constants Vs. Incubation Time

  19. In Conclusion • Succeeded in producing a DLS setup • Induced aggregation in HEWL with TFE • Unable to prove that aggregate contained Amyloid Fibrils • With more time, experiment could be completed

  20. Special Thanks Dr. Stephen Hagen Dr. Robert DeSerio Caleb Carswell University of Florida Physics Department

  21. (Applause)

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