biacore course summary
TRANSCRIPT
Biacore system application
Proteins interaction and Binding Affinity Analysis
Baijun Kou, M.D, Ph.DBaylor College of Medicine
July17-18,2012. San Diego
•Surface Plasmon resonance (SPR) detects refractive index changes close to the surface•The accumulation of 1 pg/mm2 gives a change of 1 RU
Analysis Cycle
Surface preparation
Analysis Cycle
The Steps in the Biacore Assay
Sample injection
Regeneration
Evaluation
Optimization of pH for immobilization
Immobilization of ligand to Sensor chip
Immobilization
• What to immobilize Ligand vs. analyte
• Available sensor chip surface Ligand /analyte properties to decide
• How to create a ligand surface Direct vs. capture
• Method to immobilize Amine coupling vs. others
• Control
How to create a ligand surface
Direct Capture
a n alyte
lig a n d
a n alyte
lig a n d
ca pturin g
m ole cule
The advantage of capture technique • Always a fresh surface
• Homogeneous presentation
• Regeneration conditions maybe easier to determine
• Complete purification is not necessary
Sensor surface
•Covalent chemistry
•Often heterogenous orientation
•Higher binding capacity
•Orientation-specific
•Selective ligand capture
•Lower binding capacity
Immobilization procedure
• pH scouting
Antibody in variable pH of Acetate contact with chip for 2min NaOH to remove binding
• Activation = EDC/NHS injection surface esters• Ligand contact = reaction with amine groups on ligand• Blocking = deactivation of free esters with ethanolamine
Immobilization
Activation
Blocking
Ligand contact
RL
Immobilization level
• The binding capacity of the surface(Rmax) depends on the immobilization level
• Different applications require different immobilization levels
Kinetic analysis Rmax is between 50-250RU
• Rmax describes the binding capacity of the surface
Rmax=analyte MW / ligand MW *RL*Sm RL=the immobilization level Sm= number of binding sites on ligand for analyte.
• The theoretical Rmax is often higher than the experimental Rmax
Analyte
• The relationship of Concentration, KD and Rmax
Mass transport limitation
• Mass-transfer limited binding can cause of deviation from 1:1 binding
Balance between analyte consuption and analyte supply
Overcome• Fast flow rates reduce the diffusion distance
•Low Rmax reduces the comsuption
•Mass transport correction included in all kinetic models
Regeneration
• Remove bound analyte completely from the surface• Maintain the activity of the surface• Efficient regeneration is crucial for high-quality data
• Start with mild regeneration conditions• Increase flow rate to 50-100ul/min Follow injection with Extraclean• Use short contact times with extreme
conditions• Be creative Regeneration cocltail
Strategy
Regeneration Scouting
•Efficient regeneration removes all bound analyte
•A second injection of analyte reveals whether the ligand is still fully active
•Repeated cycles of analyte and regeneration injections are required to fully assess the conditions selected
Monitor baseline!!!
Kinetics v.s Affinity
•Kinetics•How fast do things happen?-Time-dependent
•Association-how fast molecules bind
•Dissociation-how fast complexes fall apart
•Kinetics determine whether a complex forms or dissociates within a given time span
•Affinity•How strong is a complex?-Time-independent
•Affinity determines how much complex is formed at equilibrium (steady state where association balances dissociation)
Equilibrium Constants
Equilibrium dissociation constant KD
Equilibrium association constant KA
Definition
Unit [M] [M-1]
Describes Dissociation tendency
High KD = low affinity
Association tendency
High KA = high affinity
Typical range 1x10-5 – 1x10-12 1x105 – 1x1012
kd(A).(B)
(AB)=
ka ka
(AB)(A).(B) =
kd
Kinetic vs. affinity• Kinetic rate analysis
Concentration analysis- find out active concentration
Ka /kd
• Affinity analysis Ligand fishing, relative ranking KD /KA
All target sites
occupied
30 min 60 min
100 nM 1 µM
30 min 60 min
10-5103
10-4104
10-3105
10-2106
kd
[s-1]
ka
[M-1s-1]
KD 10 nM
Same affinity but different kinetic rate
Affinity analysis
• Steady state model over a range of analyte
concentration
• Analyte C=20-80% saturation
• Plot Req against C
• C at 50% saturation is KD
• Use referenc e surface
• Include zero concentration sample
• At least one concentration in duplicate
0
5
10
15
20
0 60 120
Signal [RU]
Time [s]