This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Study interaction between low-molecular-weight Study interaction between low-molecular-weight analytes and proteinsanalytes and proteins Fluorescence lifetime as the readout parameterFluorescence lifetime as the readout parameter
Model-System:
Biotinylated label with anti-biotin antibodyBiotinylated label with anti-biotin antibody
Lifetime of labeled species changes upon binding to proteinLifetime of labeled species changes upon binding to protein
Lifetime Lifetime – More robust parameter compared to intensity – More robust parameter compared to intensity –– Independent of probe concentration Independent of probe concentration
–– Unaffected by probe volume and to Unaffected by probe volume and to some extend some extend photobleaching photobleaching
Comparison With Commercially Available DyesComparison With Commercially Available Dyes
Cyanine dyes like Cy5 and Alexa 647 exhibit smaller Cyanine dyes like Cy5 and Alexa 647 exhibit smaller lifetimelifetime changes (factor < 2) upon binding to proteinschanges (factor < 2) upon binding to proteins
Seta-633 exhibits a 10-fold lifetime change upon binding Seta-633 exhibits a 10-fold lifetime change upon binding to BSA and a 6-fold change with IgGto BSA and a 6-fold change with IgG
A Race between Emission and Molecular MotionFluorescence Polarization
A Race between Emission and Molecular MotionPolarized light excitesPolarized light excitesmolecules aligned withmolecules aligned withplane of polarizationplane of polarization
flfl<<<<rotrotflfl>>>>rotrot
Excited molecules remain aligned.Excited molecules remain aligned.Fluorescence is polarized.Fluorescence is polarized.
Orientation of excited molecules Orientation of excited molecules randomizes. Fluorescence is depolarized.randomizes. Fluorescence is depolarized.
For best results lifetime For best results lifetime flfl should be in the range of should be in the range of
rotational correlation time rotational correlation time of antigen that is labeled of antigen that is labeled
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
160
180
200
220
240
260
280
300
320
340
K7-545-BSA — conjugate
Pol
ariz
atio
n [m
P]
[ANTI-BSA / K7-545-BSA]
flfl = = 26 ns26 nsSeTau-425SeTau-425
TG-404TG-404 flfl = 9 ns= 9 ns
Lifetime Lifetime flfl of the label is a measure for MW of the of the label is a measure for MW of the species that can be measured in a FPIAspecies that can be measured in a FPIA
Commercially available Lifetime Dyes:Commercially available Lifetime Dyes:Commercially available Lifetime Dyes:Commercially available Lifetime Dyes:
Fluorescent Probes will accumulate differently at Fluorescent Probes will accumulate differently at various locations in the cell various locations in the cell
Measurement ModeMeasurement Mode Qualitative or quantitative measurements. Ion-probes showing spectral shifts - radiometric measurements Are light source available?
How to Choose the Correct Fluorescent ProbeHow to Choose the Correct Fluorescent Probe
Dissociation Constant (KDissociation Constant (Kdd)) Calibration: Kd of probe is dependent on pH, temperature,
viscosity, ionic strength etc…….
Indicator FormIndicator Form Influences cell loading and distribution of the probe Salts and dextran-conjugates - microinjection, electroporation, AM-esters - passively loaded and cleaved by intracellular esterases
Seta-650-pH probe also shows Seta-650-pH probe also shows pH- sensitive lifetime changespH- sensitive lifetime changes
Phase angle changes ~ 16 degPhase angle changes ~ 16 deg between protonated and between protonated and deprotonated forms at 100 MHzdeprotonated forms at 100 MHz
FLIM pH-Measurements in Cells FLIM pH-Measurements in Cells
pH-Label applicable for FLIM pH-Label applicable for FLIM
Seta-650-pH labeled E.Coli Seta-650-pH labeled E.Coli is internalized in Cellis internalized in Cell
Inside Cell pH of certain Inside Cell pH of certain compartments is lower pH ~ 5compartments is lower pH ~ 5
pH-Sensing in Cells - Phagocytosis pH-Sensing in Cells - Phagocytosis
Fluorescence Intensity or LifetimeFluorescence Intensity or Lifetime will reflect these pH changes and will reflect these pH changes and are utilized as parameters for are utilized as parameters for visualization visualization
Y. Povrozin Y. Povrozin ISC, Kharkov,ISC, Kharkov, Ukraine Ukraine A. Tatarets A. Tatarets ISC, KharkovISC, Kharkov, Ukraine, Ukraine L. PatsenkerL. Patsenker ISC, KharkovISC, Kharkov, Ukraine and , Ukraine and
Seta BioMedicalsSeta BioMedicals, Urbana IL, USA, Urbana IL, USA