Fluorescence detected sedimentation

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Fluorescence detected sedimentation

• Applications using sedimentation velocity

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Outline

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

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Types of sedimentation
Graphs courtesy of Peter Schuck
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Sedimentation velocity
S
D
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AU-FDS opticsConfocal fluorescence optics
PMT
Laser
Spatial filter
Collimation
Dichroic filter
Dichroic mirror
Expansion
Objective lens
Fluorescent event
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Data CollectionOne radius, several rotations

• Aviv AU-FDS
• Rapid-scan absorbance

FDS
FDS
RSA
RSA
Slit assembly
Radial steps
One rotation
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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

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Simultaneous dataacquisition

• Aviv AU-FDS
• Rapid-scan absorbance

• FDS and RSA data
• Data for all cells
• Signal averaging

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AU-FDS applications
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Determining s and Mw
40 nM GFP in 100 mM KCl, 10 mM Tris, pH 8.0
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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
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Using a lipid label DPPC-NDB rsEPCR
rsEPCR DPPC 2.87 s
rsEPCR alone 2.63 s
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GFP-labeled construct in cell lysate
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Monitoring interacting systems
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Detecting trace molecule
40 nM GFP in varying concentrations of E. coli
lysate
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40 nM GFP in concentrated solutions of different
charge
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Human serum studies
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Alexa488-BSA in TBS or Human serum
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Using labeled lipid in serum4?M DPPE
Albumin
Free DPPE?
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Alexa-488 IgG in serum
500 nM Alexa in TBS or human serum
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40 nM GFP in serum
Serum albumin
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40 nM GFP rat Anti-GFP IgG
Rachel Kroe
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Kd for GFP/Anti-GFP Interaction by fluorescence
polarization

• The Kd for this interaction is 1 nM
• Sedimentation shows the interaction is 11 and
  not 11 21

Rachel Kroe
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GFP/Anti-GFP in Human serum
100 nM GFP/100 nM Anti-GFP in Serum
Rachel Kroe
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Acknowledgements
Rachel Kroe Brett Austin BITC Jeff
Hansen CAMIS Borries Demeler NIH
Edward Eisenstein NSF