Fluorescence detected sedimentation

1. Fluorescence detected sedimentation Applications using sedimentation velocity

2. Outline • Basics of sedimentation velocity • How the AU-FDS works • Applications • Examples of applications • Where it can take us

3. Types of sedimentation Graphs courtesy of Peter Schuck

4. Sedimentation velocity S D

5. AU-FDS opticsConfocal fluorescence optics PMT Laser Spatial filter Collimation Dichroic filter Dichroicmirror Expansion Objective lens Fluorescent event

6. One rotation Signal from samples Data CollectionOne radius, several rotations • Aviv AU-FDS • Rapid-scan absorbance FDS RSA FDS RSA Slit assembly Radial steps One rotation

7. Rotor timing pulse One rotation Intensity Intensity from sample 1 Data acquisition strategy • Aviv AU-FDS • Rapid-scan absorbance • Continuous light source • Acquire light intensities and rotor timing pulse • Asynchronous with rotor • Sort out the data in computer memory

8. Simultaneous dataacquisition • Aviv AU-FDS • Rapid-scan absorbance • FDS and RSA data • Data for all cells • Signal averaging 1 2 3

9. 40 nM GFP in 100 mM KCl, 10 mM Tris, pH 8.0 40 nM GFP in E.coli lysate 100 nM GFP 100 nM Anti GFP in Human serum AU-FDS applications

10. Determining s and Mw 40 nM GFP in 100 mM KCl, 10 mM Tris, pH 8.0

11. Using a labeled lipid DPPC-NBD ± rsEPCR 400 nM DPPC-NBD + rsEPCR 20 mM Tris, 100 mM NaCl and 3 mM CaCl2, pH 7.5

12. Using a lipid label DPPC-NDB + rsEPCR rsEPCR + DPPC 2.87 s rsEPCR alone 2.63 s

13. GFP-labeled construct in cell lysate

14. Monitoring interacting systems

15. Detecting trace molecule 40 nM GFP in varying concentrations of E. coli lysate

16. Repulsion STI Dextran  HEL Attraction 40 nM GFP in concentrated solutions of different charge

17. Human serum studies

18. Alexa488-BSA in TBS or Human serum

19. Using labeled lipid in serum4M DPPE Albumin Free DPPE?

20. Alexa-488 IgG in serum 500 nM Alexa in TBS or human serum

21. 40 nM GFP in serum Serum albumin

22. GFP/Anti-GFP in Human serum 100 nM GFP/100 nM Anti-GFP in Serum Rachel Kroe

23. Acknowledgements Rachel Kroe Brett Austin BITC Jeff Hansen CAMIS Borries Demeler NIH Edward Eisenstein NSF