reichert - lunch & learn presentation
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Reichert Life Science Legacy
• 15 years of SPR expertise
• >200 publications on Reichert SPR
• Cover the full spectrum of bio molecular interactions – Protein-protein – Protein-small molecule
• Ensuring success through service– SPR support staff helps researchers solve problems– Methods development, high-volume experiments, feasibility studies
A history of exceptional performance, value and support.
The Reichert SPR Advantage • Your partner every step of the way
– Unmatched customer service and support solutions– Maximum uptime drives better results
• Solve your research bottlenecks– Scalable to research and lab needs– Systems accessible to your lab
• Reliable binding, kinetics, concentration and thermodynamic data
– Helping you answer questions quantitatively
• Increase your sample flexibility– Broader application options– Robust fluidics
• Reduce your equipment and maintenance costs— Lower operating, lifetime ownership costs
Better Fluidics, Better Results
• Run sample types you wouldn’t consider on other systems• Minimize maintenance requirements and costs
– Five-year total cost of ownership is 30% lower than leading competitor
– Tubing is easy-to-access and replace
• Variable tubing sizes – From 64 to 500 m inner diameter
• Variable sample loop volumes – From 10 to 5000 L inner diameter
• Compatible with a wide range of solvents
Advantages Over Biacore
• Robust, easy-to-access fluidics system• Specialized flow cells • User-friendly software interface• Lower capital investment and lifetime
cost of ownership • Superior, on-demand support services• Unmatched baseline stability• Comparable sensitivity and noise performance
Advanced SPR Technology
• Innovative two and four channel systems– Ideal sensitivity for low molecular weight analysis
• Noise and sensitivity performance required for challenging applications
• More applications and sample types– Robust enough for crude samples,
cell lysates, aggregates
• High sample capacity – Two 96- or 384-well plates– Up to 768 samples—or any combination
of plates and vials
• Scalable to meet your needs now and later– Solutions grow as you do – Rental services available
Commitment to Service and Your Success
• Application and methods development • On-call assistance to minimize downtime• In-lab contract sample analysis for customers• Installation training, lectures and hands-on
application support• Preventative maintenance• Remote and on-site service plans• Additional consulting services
Current Customer Sampling
Reichert4SPR System • New in 2015• Increased throughput for drug discovery
– 4 channels improve the efficiency and study design flexibility
• Advanced optics, image sensing, electronics and software
• The most robust fluid handling systems– Accommodates many more sample compositions
• Reliable results and seamless data analysis• Quick method development and
programmability• Far easier to maintain than other SPR instruments
– Max uptime to drive better results
SPECIFICATIONS
Reichert4SPR Key Specs
Reichert 7500DC Specs
CompetitiveComparisons
Reichert4SPR Biacore T200
Reichert4SPR vs GE-Biacore T200
• Equivalent performance
• Reichert Advantages:
• Robust Fluidics
• Better uptime• More flexible• Affordable
Reichert4SPR Biacore T200
Reichert 2 Channel SPR vs GE-Biacore 2 Channel SPRReichert 7500DC Biacore X100
Channels 2 2Injection of Lysates, Cells, Particulates
Yes No
DIY Fluidics Yes No
Sample Capacity 768 samples 15 SamplesBaseline Noise 0.05µRIU RMS <0.3µRIU RMSBaseline Drift 0.01µRIU RMS <0.1µRIU RMS
Sensor Chip Cost $84 dextran chip $189 dextran chipSpecialized Flowcells Electrochemistry, Photochemistry,
Flourescence, MALDI MS plugnone
Sample Volume 1-4500µL 20-30µL
Temperature Range 10oC below ambient to 70oC 10oC below ambient to 45oCSample Compartment Temperature
4oC or Ambient None
Flow Rate Range 0.1-3000µL/min 1-100µL/minMin Molecular Weight <100 Da 100 Da
SENSOR CHIPS
Reichert Sensor Surfaces (I)
• Low Binding Capacity• No Matrix Depth• Large/Moderate MW
Molecules• Low Non-Specific
Binding
• High Binding Capacity• Flexible• Large/Moderate or Low
MW Molecules• Minimal Non-Specific
Binding
= COOHCarboxyl Surfaces
Reichert Sensor Surfaces (II)
• Steptavidin Amine Coupled• Capture Biotinylated Ligands• Stable Surface
• Alkyl Surface• Capture Lipid Monolayer• Regenerate with CHAPS
• Nitriloacetic Acetic Acid Surface• Capture HIS-Tagged Ligands• Decaying Surface• Regenerate with Imidazole
• Protein A Amine Coupled• Capture IgGs• Regenerate with pH 2 buffer
SPR TECHNOLOGY
Surface Plasmon Resonance
Metal SurfacePlasmons (Electron Waves)
Surface Plasmon Resonance AngleIn
tens
ityDetecto
r
780
nm L
ight
> qc Resonance (Energy Transfer)
Surface Plasmon Resonance
Angle
Inte
nsity
Change in Refractive Index
Changes in Refractive Index
Angle
Inte
nsity
Mass Sensor
Detecto
r
Angle
Inte
nsity
Time
Res
pons
e
Raw Data
Response Data
780
nm L
ight
Response Units
Time
Res
pons
e
Units are in RIU (10-6 refractive index units)
1 RIU = 1 pg/mm2 of mass binding
A very precise refractometer
A very precise mass sensor
Versatile Technique
Time
Res
pons
e
Binding Reponse
Baseline
Is there an interaction? (Yes/No Binding)
How strong is the interaction? (Affinity)
How quickly do they interact and dissociate? (Kinetics)
Why? (Thermodynamics) (DH, DS, DG)
How much? (Concentration)
Asso
ciatio
nDissociation
Regeneration
All Classes of Biomolecules
<100 Da to Proteins to Cells
• Proteins• Lipids• Carbohydrates• Nucleic Acids
• LMW Molecules
• Whole Cells • Bacteria, Viruses
The Importance of Kinetics
Series1
0.0001 0.001 0.01 0.1 1102
103
104
105
106
107
kd (s-1)
k a (M
-1s-1
)
10 pM 100 pM 1 nM 10 nM
1 M
10 M
100 M
1 mM
100 nM
KD
KD(M) = kd (s-1)ka (M-1s-1)
Conventional Regeneration Approach
SampleReference
17900
18100
18300
18500
18700
18900
1000 2000 3000 4000 5000 6000
Time (sec)
Res
pons
e (
RIU
)
• Each concentration in a single cycle• Requires regeneration between
injections
Equilibrium Data
KD = 940 nM
Measuring Responses at Equilibrium
Steady-State Measurement - not Kinetics
Another way to measure KD
aside from kinetics
Thermodynamics
0
20
40
60
80
0 50 100 150 200 250
Time (sec)
Resp
onse
(uRI
U)
Time (sec)
Res
pons
e (u
RIU
)
20 oC
35 oC
Measure Binding at Different Temperatures (at least 5)
Van’t Hoff Equation
Van’t Hoff Plot
Slope = DHR
-14.4
-13.8
-13.2
-12.63.2 3.3 3.4 3.5
1/T (X 10-3 K-1)
ln (K
D)
Y-Intercept = DSR
Concentration Determination
Determine Concentration of Biomolecule in Solution
Surface with Specific Antibody
APPLICATIONS
•Anti-HSA IgG / HSA•Enzyme-Inhibitor Interactions•Lipid-Peptide Interactions•Carbohydrate-Receptor Interactions•Development of a Cytokine Antagonist•Cell-Protein Binding
APPLICATION 1: ANTI-HSA IGG/HSA
Ligand: Anti-HSA IgGAnalyte: Human Serum Albumin (HSA)
Amine Coupling
animation
animation
Anti-HSA Immobilization
10000
15000
20000
25000
30000
35000
0 500 1000 1500 2000 2500 3000 3500 4000 4500
Time (sec)
Res
pons
e (u
RIU
) EDC/NHS
Anti-HSA
1M Ethanolamine, pH 8.5
HSA Injections
Sample
Reference
17900
18100
18300
18500
18700
18900
1000 2000 3000 4000 5000 6000
Time (sec)
Res
pons
e (
RIU
)
HSA Kinetic Results
APPLICATION 2: ENZYME-INHIBITOR INTERACTIONS
Ligand: Carbonic Anhydrase IIAnalytes: 4-Carboxybenzene Sulfonamide (200 Da) and Methane Sulfonamide (95 Da)
Inhibitor Kinetics4-CBS
Methane Sulfonamide
KD = 857 nM
KD = 650 M
APPLICATION 3: LIPID-PEPTIDE INTERACTIONS
Hydrophobic Sensor ChipPrepare Liposome SurfaceInject Peptide
Vesicle CapturePS Vesicles Capture on Phytosphingosine Surface
10000
10500
11000
11500
12000
12500
13000
13500
14000
14500
15000
10550 11550 12550 13550 14550 15550
Time (sec)R
espo
nse
(uR
IU)
Sample ChannelReference Channel
3260 uRIU of PS Vesicles on surface
• Lipophilic surface preserves vesicle bilayer conformation
• QCM dissipation data verifies intact vesicles
Phosphorylated Protein
KD = 3.92 nM
KD = 467 pM
APPLICATION 4: CARBOHYDRATE-RECEPTOR INTERACTIONS
Carbohydrate-Receptor Interactions
• Direct SPR assay to investigate carbohydrate-lectin binding kinetics
• Concanavalin A (Con A) and mannose derivatives used as a model
• Gain insight into the fundamental mechanisms of multivalent carbohydrate binding
• Methyl-a-D-mannopyranoside used as a control
• Three clicked mannosylated GATC dendrimers
Eva Maria Munoz; Juan Correa; Eduardo Fernandez-Megia; Ricardo Riguera. JACS 2009, 131, 17765-17767.
Sensorgrams
• Monosaccharide/Con A binding is comprised of fast on and off rates that follow a 1:1 binding model
• Glyco-dendrimers have more complex binding kinetics consistent with the multivalent nature of the dendrimers and the clustered arrangement of lectin
• Association rates are similar but dissociation rates varied with the generation of dendrimer
Eva Maria Munoz; Juan Correa; Eduardo Fernandez-Megia; Ricardo Riguera. JACS 2009, 131, 17765-17767.
Binding Mechanism Unraveled
• Three-phase binding model proposed:1) A low affinity binding site similar to the monosaccharide2) A higher affinity binding mode resulting from dendrimer binding with higher functional valency3) As dissociation occurs from the low affinity binding site, the sites become occupied by higher
functional valency dendrimers.
Eva Maria Munoz; Juan Correa; Eduardo Fernandez-Megia; Ricardo Riguera. JACS 2009, 131, 17765-17767.
APPLICATION 5: DEVELOPMENT OF A CYTOKINE ANTAGONIST
Design of the Antagonist
• IL-1 Cytokines are master mediators of the inflammatory response.
• IL-1α and IL-1β protein are elevated in the lacrimal gland, tears, and the ocular surface in all forms of dry-eye disease
• Chimerized two IL-1 receptor ligands, IL-1β and IL-1Ra, to create an optimized receptor antagonist.
Jinzhao Hou, et.al. PNAS 2013, 110, 3913-3918.
Jinzhao Hou, et.al. PNAS 2013, 110, 3913-3918.
SPR Sensorgrams
SPR Results
• Focused on chimera 93:60
Jinzhao Hou, et.al. PNAS 2013, 110, 3913-3918.
APPLICATION 6: CELL-PROTEIN BINDING
Human Embryonic Kidney
HEK Cells Capture
200 nM Fibrinogen Injection
Surface Heterogeneity Model
ka1 = 2.40 e3 M-1s-1
kd1 = 8.51 e-3 s-1
KD1 = 3.54 M
ka2 = 9.74 e3 M-1s-1
kd2 = 2.57 e-4 s-1
KD2 = 26.4 nM
SPECIALTY APPLICATIONS
•Electrochemistry
•Mass Spectrometery
•Photochemistry/Fluorescence
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