Fluorescence detected sedimentation Applications using sedimentation velocity Page

  • Slides: 23
Download presentation
Fluorescence detected sedimentation Applications using sedimentation velocity Page

Fluorescence detected sedimentation Applications using sedimentation velocity Page

Outline • Basics of sedimentation velocity • How the AU-FDS works • Applications •

Outline • Basics of sedimentation velocity • How the AU-FDS works • Applications • Examples of applications • Where it can take us 2 10/3/2020

Types of sedimentation Graphs courtesy of Peter Schuck

Types of sedimentation Graphs courtesy of Peter Schuck

Sedimentation velocity S D 4 10/3/2020

Sedimentation velocity S D 4 10/3/2020

AU-FDS optics Confocal fluorescence optics Spatial filter Collimation Laser PMT Dichroic filter Dichroic mirror

AU-FDS optics Confocal fluorescence optics Spatial filter Collimation Laser PMT Dichroic filter Dichroic mirror Objective lens Expansion Fluorescent event 5 10/3/2020

Data Collection • • One radius, several rotations Aviv AU-FDS Rapid-scan absorbance FDS RSA

Data Collection • • One radius, several rotations Aviv AU-FDS Rapid-scan absorbance FDS RSA Slit assembly One rotation Radial steps Signal from samples 6 One rotation 10/3/2020

Data acquisition strategy • • Aviv AU-FDS Rapid-scan absorbance • Continuous light source •

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 Rotor timing pulse One rotation Intensity from sample 1 7 10/3/2020

Simultaneous data acquisition • • Aviv AU-FDS Rapid-scan absorbance • FDS and RSA data

Simultaneous data acquisition • • Aviv AU-FDS Rapid-scan absorbance • FDS and RSA data • Data for all cells • Signal averaging 1 8 2 3 10/3/2020

AU-FDS applications 40 n. M GFP in 100 m. M KCl, 10 m. M

AU-FDS applications 40 n. M GFP in 100 m. M KCl, 10 m. M Tris, p. H 8. 0 100 n. M GFP 100 n. M Anti GFP in Human serum 40 n. M GFP in E. coli lysate 9 10/3/2020

Determining s and Mw 40 n. M GFP in 100 m. M KCl, 10

Determining s and Mw 40 n. M GFP in 100 m. M KCl, 10 m. M Tris, p. H 8. 0 10 10/3/2020

Using a labeled lipid DPPC-NBD ± rs. EPCR 400 n. M DPPC-NBD + rs.

Using a labeled lipid DPPC-NBD ± rs. EPCR 400 n. M DPPC-NBD + rs. EPCR 20 m. M Tris, 100 m. M Na. Cl and 3 m. M Ca. Cl 2, p. H 7. 5 11 10/3/2020

Using a lipid label DPPC-NDB + rs. EPCR + DPPC 2. 87 s 12

Using a lipid label DPPC-NDB + rs. EPCR + DPPC 2. 87 s 12 rs. EPCR alone 2. 63 s 10/3/2020

GFP-labeled construct in cell lysate 13 10/3/2020

GFP-labeled construct in cell lysate 13 10/3/2020

Monitoring interacting systems 14 10/3/2020

Monitoring interacting systems 14 10/3/2020

Detecting trace molecule 40 n. M GFP in varying concentrations of E. coli lysate

Detecting trace molecule 40 n. M GFP in varying concentrations of E. coli lysate 15 10/3/2020

Repulsion 40 n. M GFP in concentrated solutions of different charge Dextran Attraction 16

Repulsion 40 n. M GFP in concentrated solutions of different charge Dextran Attraction 16 STI HEL 10/3/2020

Human serum studies Page

Human serum studies Page

Alexa 488 -BSA in TBS or Human serum 18 10/3/2020

Alexa 488 -BSA in TBS or Human serum 18 10/3/2020

Using labeled lipid in serum 4 M DPPE Albumin Free DPPE? 19 10/3/2020

Using labeled lipid in serum 4 M DPPE Albumin Free DPPE? 19 10/3/2020

Alexa-488 Ig. G in serum 500 n. M Alexa in TBS or human serum

Alexa-488 Ig. G in serum 500 n. M Alexa in TBS or human serum 20 10/3/2020

40 n. M GFP in serum Serum albumin 21 10/3/2020

40 n. M GFP in serum Serum albumin 21 10/3/2020

GFP/Anti-GFP in Human serum 100 n. M GFP/100 n. M Anti-GFP in Serum 24

GFP/Anti-GFP in Human serum 100 n. M GFP/100 n. M Anti-GFP in Serum 24 Rachel Kroe 10/3/2020

Acknowledgements Rachel Kroe Brett Austin BITC CAMIS NIH NSF 25 Jeff Hansen Borries Demeler

Acknowledgements Rachel Kroe Brett Austin BITC CAMIS NIH NSF 25 Jeff Hansen Borries Demeler Edward Eisenstein 10/3/2020

3 3 Velocity and Acceleration Velocity Average Velocity3 3 Velocity and Acceleration Velocity Average Velocity
Relative velocity Contents Relative velocity equation Relative velocityRelative velocity Contents Relative velocity equation Relative velocity
Velocity and Acceleration Kinematics 1 Velocity Velocity isVelocity and Acceleration Kinematics 1 Velocity Velocity is
Fluorescence for fluorophobesVirtual fluorescence using chromagens and histochemicalFluorescence for fluorophobesVirtual fluorescence using chromagens and histochemical
6 1 Configuration of sedimentation tank Sedimentation is6 1 Configuration of sedimentation tank Sedimentation is
SEDIMENTATION Outline General Theory Types of sedimentation basinSEDIMENTATION Outline General Theory Types of sedimentation basin
Sedimentation Sedimentation is a physical treatment process thatSedimentation Sedimentation is a physical treatment process that
Erythrocyte Sedimentation Rate n n n Erythrocyte sedimentationErythrocyte Sedimentation Rate n n n Erythrocyte sedimentation
Sedimentation Biophysical Tools 02 Sedimentation 1192020 1 PrincipleSedimentation Biophysical Tools 02 Sedimentation 1192020 1 Principle
Erythrocyte Sedimentation Rate ESR Erythrocyte Sedimentation Rate ItErythrocyte Sedimentation Rate ESR Erythrocyte Sedimentation Rate It
Performing a Erythrocyte Sedimentation Rate Erythrocyte Sedimentation RatePerforming a Erythrocyte Sedimentation Rate Erythrocyte Sedimentation Rate
Sedimentation Sedimentation is a physical treatment process thatSedimentation Sedimentation is a physical treatment process that
Design of Primary Sedimentation Tank A primary sedimentationDesign of Primary Sedimentation Tank A primary sedimentation
Sedimentation Biophysical Tools 02 Sedimentation 9172021 1 PrincipleSedimentation Biophysical Tools 02 Sedimentation 9172021 1 Principle
Separation Techniques Method 1 Sedimentation Sedimentation is theSeparation Techniques Method 1 Sedimentation Sedimentation is the
SEDIMENTATION 12272021 1 Definition Sedimentation is the tendencySEDIMENTATION 12272021 1 Definition Sedimentation is the tendency
SEDIMENTATION 12212021 1 Definition Sedimentation is the tendencySEDIMENTATION 12212021 1 Definition Sedimentation is the tendency
CE 370 Sedimentation Sedimentation Objectives l Uses lCE 370 Sedimentation Sedimentation Objectives l Uses l
Sedimentation Lesson five Reducing Sedimentation Based on yourSedimentation Lesson five Reducing Sedimentation Based on your
Spectroscopie de Fluorescence Sparation des pics de fluorescenceSpectroscopie de Fluorescence Sparation des pics de fluorescence
7 Fluorescence microscopy 7 5 Fluorescence Recovery After7 Fluorescence microscopy 7 5 Fluorescence Recovery After
Fluorescence Polarization Immunoassay FPIA Fluorescence polarization is idealFluorescence Polarization Immunoassay FPIA Fluorescence polarization is ideal