Molecular interactions observed in a real time
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Transcript of Molecular interactions observed in a real time
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Molecular interactions observed in a real time
Bogdan WalkowiakDepartment of Biophysics
Technical University of Lodz
and
Department of Molecular and Medical Biophysics, Medical University of Lodz
– possibilities and limitations of
SPR technology
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Endpoint assay
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Introducing a binding profile
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TIR – Total Internal Reflection
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SPR – Surface Plasmon Resonanse
kx=2n1sin(/
ksp=(ng2n2
2/ng2+n2
2)0.5
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SPR Detection System
Sensor chip withgold film
Prism
Flow channel
Light source
Polarizedlight
Optical detection
unit
Reflected light
III
I II
II
I
Sensorgram
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Microfluidic system IFC
Liquid Handling
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Gold-Dextran Surfaces
• Biocompatible
• Low non-specificbinding
• More than 100 runs on the same surface
Specific layer
Dextran layer
Linker layer
Gold film
Glass
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The Sensorgram
ResonanceSignal (KRU)
Dissociation
Regeneration
Ass
oci
atio
n Kinetics
Concentration
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Time (s)
18
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BiaCore sensor
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BiaCore sensor family
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Correlation between SPR response and surface concentration
Signal proportional to massSame specific response for different proteins
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time (s)
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.)
pH 6.8
pH 4.0
pH 2.8
Protein concentration at the sensor surface
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Coupling Chemistries
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Protein immobilization at the sensor surface
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time (s)
resp
on
se (
R.U
.)
activation
concentration andimmobilization
saturation
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.U.)
fibrin, Sta K2, 25 ug/ml
fibrin, Staphylo, 25 ug/ml
dextran, Sta K2, 25 ug/ml
dextran, Staphylo, 25 ug/ml
Szemraj J., Pietrucha T., Nowak P., Walkowiak B. In preparation 2002
Recombinant protein mediating fibrynolyitc process
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.)
pH 8,2
pH 8,4
pH 8,0
pH-dependence of MBP and DNAK interaction
Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002
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pH
resp
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.)
Final presentation of pH-dependence for MBP and DNAK
Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002
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.)
specif ic antibody binding to MBP protein
nonspecif ic binding
Specific antibody binding to immobilized MBP protein
Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002
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DNAK protein binding to MBP proteinDNAK protein binding to MBP protein in the presence of specific antibody
Specific binding inhibition by Mab against MBP protein
Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002
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Acid glycoprotein binding to immobilized PAI-1
Boncela J., Papiweska I., Fijałkowska I., Walkowiak B., Cierniewski CS. J..Biol.Chem,276, 35305, 2001
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Vitronectin binding to immobilized PAI-1
Boncela J., Papiweska I., Fijałkowska I., Walkowiak B., Cierniewski CS. J..Biol.Chem,276, 35305, 2001
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Glass plate one side covered with gold film
Carbon deposition by RF CVPD technology
Preparation of sensor surface for NCD-plasma proteins interaction
Walkowiak B et.al. J. Wide Bandgap Materials, June 2002
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R (1/cm)
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a.u
.)NCD surface
gold surface
Raman spectrometry analysis of prepared sensor
surface
Walkowiak B et.al. J. Wide Bandgap Materials, June 2002
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urfa
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.U.)
gold surface
NCD surface
platelet poor plasma rabbit serum anti-fg
plasma proteins adsorbed on the sensor surface
antibodies bound to fibrinogen
Adsorbtion of plasma proteins to the NCD surface
Walkowiak B et.al. J. Wide Bandgap Materials, June 2002
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Sensor for detection of platelet adhesion
to immobilized fibrinogen
Immobilization of rabbit anti-fibrinogen at the sensor surface
Walkowiak B et.al. J. Wide Bandgap Materials, June 2002
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.)
fibrinogen suspension of blood platelets
bound fibrinogen
adheredplatelets
regeneration
Adhesion of blood platelets to the sensor with immobilized fibrinogen
Walkowiak B et.al. J. Wide Bandgap Materials, June 2002
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Cells interaction with immobilized ligands
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resp
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.)140 mM NaCl
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Effect of high salt concentration on antibody-ligand interaction
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Effect of high salt concentration on antibody-ligand interaction
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.)140 mM NaCl
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Sample Recovery and Fractionation
563
Fractionate dissociating analyte for- re-injection
- sequencing
- mass spectrometry
- electrophoresis
- PCR
Recover eluted material for- enzymatic transfer assays
- on-surface cleavage processes
- alterations due to binding or regeneration
Recover excess sample during injection
- save expensive sample and reagents
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BiaCore - MS
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Limitations
Molecular weight of analyte not less than 200
Limitet chemical resistivity to organic solvents
Residual interaction of analyte with dextran matrix
Active for measuremet distance from a sensorsurface is limited to 300 nm
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Thank you very much
for your attention