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Similaire à Reichert SPR Legacy of Excellence
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Reichert SPR Legacy of Excellence
- 2. 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.
- 3. 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
- 4. 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
- 5. 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
- 6. 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
- 7. 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
- 9. 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
- 10. SPECIFICATIONS
- 14. Reichert4SPR Biacore T200 Reichert4SPR vs GE-Biacore T200 • Equivalent performance • Reichert Advantages: • Robust Fluidics • Better uptime • More flexible • Affordable Reichert4SPR Biacore T200
- 15. Reichert 2 Channel SPR vs GE-Biacore 2 Channel SPR Reichert 7500DC Biacore X100 Channels 2 2 Injection of Lysates, Cells, Particulates Yes No DIY Fluidics Yes No Sample Capacity 768 samples 15 Samples Baseline Noise 0.05µRIU RMS <0.3µRIU RMS Baseline Drift 0.01µRIU RMS <0.1µRIU RMS Sensor Chip Cost $84 dextran chip $189 dextran chip Specialized Flowcells Electrochemistry, Photochemistry, Flourescence, MALDI MS plug none Sample Volume 1-4500µL 20-30µL Temperature Range 10oC below ambient to 70oC 10oC below ambient to 45oC Sample Compartment Temperature 4oC or Ambient None Flow Rate Range 0.1-3000µL/min 1-100µL/min Min Molecular Weight <100 Da 100 Da
- 16. SENSOR CHIPS
- 17. 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 = COOH Carboxyl Surfaces
- 18. 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
- 19. SPR TECHNOLOGY
- 20. Surface Plasmon Resonance Metal Surface Plasmons (Electron Waves) Surface Plasmon Resonance Angle Intensity > qc Resonance (Energy Transfer)
- 22. Change in Refractive Index Changes in Refractive Index Angle Intensity
- 24. Response Units Time Response 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
- 25. Versatile Technique Time Response 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) Regeneration
- 26. All Classes of Biomolecules <100 Da to Proteins to Cells • Proteins • Lipids • Carbohydrates • Nucleic Acids • LMW Molecules • Whole Cells • Bacteria, Viruses
- 27. The Importance of Kinetics 0.0001 0.001 0.01 0.1 1 102 103 104 105 106 107 kd (s-1) ka(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)
- 28. Conventional Regeneration Approach Sample Reference 17900 18100 18300 18500 18700 18900 1000 2000 3000 4000 5000 6000 Time (sec) Response(RIU) • Each concentration in a single cycle • Requires regeneration between injections
- 29. Equilibrium Data KD = 940 nM Measuring Responses at Equilibrium Steady-State Measurement - not Kinetics Another way to measure KD aside from kinetics
- 30. Thermodynamics 0 20 40 60 80 0 50 100 150 200 250 Time (sec) Response(uRIU) Time (sec) Response(uRIU) 20 oC 35 oC Measure Binding at Different Temperatures (at least 5) Van’t Hoff Equation
- 31. Van’t Hoff Plot Slope = DH R -14.4 -13.8 -13.2 -12.6 3.2 3.3 3.4 3.5 1/T (X 10-3 K-1) ln(KD) Y-Intercept = DS R
- 32. Concentration Determination Determine Concentration of Biomolecule in Solution Surface with Specific Antibody
- 33. APPLICATIONS
- 34. •Anti-HSA IgG / HSA •Enzyme-Inhibitor Interactions •Lipid-Peptide Interactions •Carbohydrate-Receptor Interactions •Development of a Cytokine Antagonist •Cell-Protein Binding
- 35. APPLICATION 1: ANTI-HSA IGG/HSA Ligand: Anti-HSA IgG Analyte: Human Serum Albumin (HSA)
- 37. Anti-HSA Immobilization 10000 15000 20000 25000 30000 35000 0 500 1000 1500 2000 2500 3000 3500 4000 4500 Time (sec) Response(uRIU) EDC/NHS Anti-HSA 1M Ethanolamine, pH 8.5
- 38. HSA Injections Sample Reference 17900 18100 18300 18500 18700 18900 1000 2000 3000 4000 5000 6000 Time (sec) Response(RIU)
- 40. APPLICATION 2: ENZYME- INHIBITOR INTERACTIONS Ligand: Carbonic Anhydrase II Analytes: 4-Carboxybenzene Sulfonamide (200 Da) and Methane Sulfonamide (95 Da)
- 41. Inhibitor Kinetics 4-CBS Methane Sulfonamide KD = 857 nM KD = 650 M
- 42. APPLICATION 3: LIPID-PEPTIDE INTERACTIONS Hydrophobic Sensor Chip Prepare Liposome Surface Inject Peptide
- 43. Vesicle Capture PS Vesicles Capture on Phytosphingosine Surface 10000 10500 11000 11500 12000 12500 13000 13500 14000 14500 15000 10550 11550 12550 13550 14550 15550 Time (sec) Response(uRIU) Sample Channel Reference Channel 3260 uRIU of PS Vesicles on surface • Lipophilic surface preserves vesicle bilayer conformation • QCM dissipation data verifies intact vesicles
- 44. Phosphorylated Protein KD = 3.92 nM KD = 467 pM
- 46. 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.
- 47. 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.
- 48. Binding Mechanism Unraveled • Three-phase binding model proposed: 1) A low affinity binding site similar to the monosaccharide 2) A higher affinity binding mode resulting from dendrimer binding with higher functional valency 3) 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.
- 49. APPLICATION 5: DEVELOPMENT OF A CYTOKINE ANTAGONIST
- 50. 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.
- 51. Jinzhao Hou, et.al. PNAS 2013, 110, 3913-3918. SPR Sensorgrams
- 52. SPR Results • Focused on chimera 93:60 Jinzhao Hou, et.al. PNAS 2013, 110, 3913-3918.
- 53. APPLICATION 6: CELL-PROTEIN BINDING Human Embryonic Kidney
- 55. 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