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Transcript of 42715699
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Characterisation of Molecular Interactions
Using Surface Plasmon Resonance: BIAcore
Nathan Scott
Wednesday 21stMay
Technique Workshop: Protein-Ligand Interactions
Centre for Structural Biology
Department of Life Sciences
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Overview
Scope of the technique
Principle of SPR
Application
Advantages and Limitations
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Molecular Interactions
Interactomics
Signal
Transduction
Cell Adhesion
Antibody Binding
Endogenous
Receptor-Ligand
Interactions
DNA BindingProteins
DrugTarget
Interactions
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What can be studied?
Scope of
Technique
Proteins
Nucleic AcidsLipids &
membrane
molecules
Carbohydrates
Small-Molecule
Drugs
Whole Cells
Viruses Bacteria
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Biomolecular Interaction Analysis: BIAcore
Detection principle: Surface Plasmon Resonance: SPR
One binding partner (LIGAND) immobilised on chip
Other (ANALYTE) injected: microfluidics
PC collects binding data in real time
Chip is regenerated to remove analyte
Cycle is repeated
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Comprehensive information
Molecular interactions in real time:
Detect
Yes/No
Identify
Specificity
Binding partners
Characterize
Affinity
Kinetics
Epitope mapping
Concentration
Thermodynamics
Mass Spec Link-Up
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Versatility
Range of chip surfaces
Range of coupling chemistries for different needs
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Surface Plasmon Resonance
Critical angle of polarised light total internal reflection
Results in excitation of surface plasmons in the gold
Creates evanescent wave fielddissipates into sample matrix
Intensity of reflected light depletes
Critical angle changes with changes in sample matrix (binding)
Change in angle is converted to resonance signal directly proportional to mass bound at surface
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Case Study
Antibody Kinetics
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Kinetics provides unique information
Two molecules with
identical affinity
Kinetic profiling differssignificantly
Cannot be seen with
end-point assays
Physiological relevance
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Antibody Engineering
Molecular Engineering
Whole IgGWhole IgG
Single-Chain Fv
VHVL
Retains monovalent binding affinity
Improved biodistribution/tissue penetration
Can be manipulated significantly
Can be selected from combinatorial libraries
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AssayFLOW
100mM Sodium Phosphate + 300mM NaCl + Surfactant p20 (0.005%) + Azide (0.005%) pH 7.4
Streptavidin
Chip
Platform (SA)
Gold surface
Carboxymethylated
dextran
Streptavidin
Optical Interface
10mM Glycine-HCl (pH 2.5)
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Toxin Immbolisation and
Stability Assessment
-100
0
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300
400
500
600
700
800
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1000
0 500 1000 1500 2000
sTime
RU
Response
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700
800
900
1000
1100
1200
0 100 200 300 400 500 600 700 800
Time s
Resp.Diff.
RU
Testing scFv Binding & Regeneration
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Testing scFv Binding & Regeneration
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When there are problems....
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
0 1000 2000 3000 4000 5000 6000
Time s
Resp.Diff.
RU
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BIA-Evaluation Software
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Results
Parameter Value
KD 136nM
koff 1.45 x 10-03 S-1
kon 1.07 x 104 M-1S-1
T-value (koff) 120
T-value (kon) 3.6 x 103
Chi-Squared 1.07
Model: Langmuir 1:1
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So you want to play on the BIAcore
Express & Purify
Your Ligand/AnalyteLabel if necessary
Dialyse/exchange
into
appropriate buffer
Immobilise ligand
on chip
- Be careful!!
Determine optimal
regeneration buffer
Dissociate your
ligand/analyte
without damaging
or removing ligand
Kinetics
Studies
Concentration Determination
Specificity
Equilibrium Studies
Check binding
by ELISA*
What buffers are
both your analyte
& ligand stable in?
Immobilisation buffer
may be different
From running buffer
Running buffer
should
contain a surfactant
Optimise buffer and
amount of ligand
Kinetics Experiment!!
LOW
HIGH
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Contacts and Booking
Dr Lisa Jennings: Application Specialist GE Healthcare
E-mail: [email protected]
Book the BIAcore at: www3.imperial.ac.uk/cmmi/core
Training strongly advised before using machine
Onsite (Discounted):
Contact Prof. Kurt Drickamer; E-mail: [email protected]
Will be a waiting time until enough people for course to take place
Externally (Cambridge):
Book at www.biacore.com
mailto:[email protected]:[email protected]://www.biacore.com/http://www.biacore.com/mailto:[email protected]:[email protected] -
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Acknowledgements
Wellcome Trust
Dr Mahendra Deonarain
All the R.A.T lab members!
Dr Lisa Jennings