Basic Biacore Course. Introduction to SPR technology. Biacore Training

Transcription

1 Basic Biacore Course Introduction to SPR technology

2 What Biacore measure Biomolecular Interactions Specificity Kinetics How Specific... How Fast & How Strong... Affinity Concentration Thermodynamics How Much... Why fast, why strong...

3 The Corner Stones of the Technology SPR Detection System Sensor Chips IFC Microfluidic System

4 #1 : Surface Plasmon Resonance principle

5 SPR detection SPR is a refractive index sensor Measurements are dependent on surface concentration & temperature SPR response is a measure of changes in the resonance angle As a rough approximation, a response of 1 RU is equivalent to change in a surface concentration of about 1 pg/mm 2 (for proteins on Sensor Chip CM5).

6 #2 : Microfluidics Liquid handling Miniaturized system Low volume of reagents Integrated and automated liquid handling IFC (expanded view) flow cells

7 Monitoring interactions with Biacore systems second interaction partner flows over sensor surface Response (RU) One interaction partner immobilized on sensor surface Sensorgram interaction on sensor surface baseline change in mass concentration proportional change in refractive index at sensor surface detected in real-time using phenomenon of surface plasmon resonance (SPR) Time association dissociation any remaining sample removed by regeneration - surface is ready for next analysis cycle buffer sample buffer

8 BIA Terminologies Ligand : Bound component Analyte: Flow-through component

9 SPR Sensing Measuring at the Surface Prism Sensitivity Metal film Dextran + immobilized protein - the hydrogel Distance [nm]

10 General Steps of a Biacore Assay Surface preparation Sample injection Regeneration Evaluation

11 #3: Sensor Chip Technology Dextran CM5 hydrophilic for coupling Linker layer Gold 50 nm for SPR Glass support Stability Dextran matrix is present on all surfaces except HPA, Au and C1

12 The Dextran Matrix Hydrophilic Flexible Resembles a 2% aqueous dextran solution environment Low non-specific binding High binding capacity Easy to activate and use for covalent coupling Withstands extensive regeneration

13 Sensor Chip CM5 Carboxymethylated dextran matrix The most versatile chip available Excellent chemical stability and assay reproducibility

14 Sensor Chips for a multitude of molecules Immobilization of interaction partners:» Proteins» Tagged proteins (His, biotin)» LMW molecules» Membrane associated molecules» Nucleic acids» Carbohydrates» Viruses or intact cells

15 Sensor Chip surfaces A summary CM5: The most versatile chip CM4: Lower degree of carboxylation (30% of CM5). May help reducing nonspecific binding in e.g. crude sample environments CM3: Shorter dextran. Same degree of carboxylation like CM5. For low immobilization levels and work with cell and viruses C1: No dextran. Flat carobxylmethylated surface. For work with cells and particles and when dextran matrix is not needed SA: For capture of biotinylated ligands NTA: For capture of His-tagged ligands HPA: For looking at lipid monolayers interacting with membrane binding biomolecules L1: For capture of liposomes with retained lipid bilayer structure

16 Information in a sensorgram

17 General Steps of a Biacore Assay Surface preparation Sample injection Regeneration Evaluation

18 General Steps of a Biacore Assay Surface preparation Sample injection Regeneration Evaluation

19 Immobilization What is immobilization?» Covalent linking of a ligand molecule to the sensor surface Points to consider» What to immobilize?» Coupling methods» Basic procedure» How much to immobilize?» Controlling the process

20 What to Immobilize The Strong and the Small strong = withstand regeneration small = the greatest signal-to-noise ratio Other Properties to Consider:» purity at least 95%by silver stain PAGE» limiting reagent» specific attachment point» component maintains functionality

21 Coupling Methods Direct immobilization» Covalent coupling to surface» Chemistries include amine, thiol & aldehyde Capture immobilization» A capture molecule is covalently coupled to the surface and captures ligand during each cycle via a high-affinity interaction» Techniques for capture include streptavidin-biotin, anti-gst/gst & RAMFc-MAb a n a lyte lig an d ana lyte lig and ca pturing m olecule

22 Coupling Chemistries

23 Basic Steps Preconcentration Activation Ligand Immobilization Blocking

24 The isoelectric point (pi) of the protein Defined as the ph at which there is no net charge of the protein ph<pi: The net charge of the protein will be positive ph>pi: The net charge of the protein will be negative

25 Pre-concentration Ligand is concentrated at the sensor surface by electrostatic attraction ligand isolectric point pi ph < 3.5 surface pk 3.5 a ligand isolectric point pi» Efficient pre-concentration requires that the ph lies between the pk a of the surface and the isoelectric point of the ligand.» Low ionic buffer strength is also important 3.5 < ph < pi ph > pi surface pk 3.5 a» Poor pre-concentration can be partially compensated for by increasing ligand concentration ligand isolectric point pi surface pk 3.5 a

26 Immobilization ph-scouting (1) The experimental procedure of finding the appropriate immobilization ph At ph>3.5 the dextran matrix carries a net negative charge Immobilization buffer ph should be higher than 3.5, but lower than the isoelectric point of the ligand For many proteins, 10 mm sodium acetate buffer (ph 4.5) works well

27 Preconcentration RU ph 5.2 Response ph mM NaOH ph Time s take advantage of electrostatic effects note slope of curve note amount nonspecifically bound

28 Activation/Coupling/Blocking RU Immobilization of anti-tsh MAb Blocking Response Activation Time s Activation = 1:1 NHS/EDC mixture Ligand Contact Ligand Contact = ligand in low ionic strength/low ph buffer Blocking = neutralize free reactive esters with amines link

29 R max R max = (MW A /MW L ) * R L * S m

30 Exercise Calculation of R L analytemw ligand MW R max = RL S m How much ligand should I immobilize if I want an R max of 100 RU? analyte MW = 25,000 Da ligand MW = 150,000 Da S m = 1 R max = 100 RU Answer: R L =600 RU [Ligand] : 2-200nM (ug/ml)

31 General Steps of a Biacore Assay Surface preparation Sample injection Regeneration Evaluation

32 Know Your Analyte Buffer» match running and sample buffers to reduce bulk effects Purity and Solubility» is the sample homogenous?» does the biomolecule aggregate easily? Information from other techniques» incorporate into experimental design» useful in data interpretation Concentration range to use» establish specificity» test with high analyte concentration» kinetic and affinity experiments» use a range of concentrations Analyte concentration: 1-100nM

33 BIA Terminologies Bulk Responses SPR responses caused by the difference in bulk refractive index between the sample and the running buffer

34 Binding and Bulk Effects Binding + Bulk Buffer 1 Bulk Buffer 1 Bulk effects are due to differences in the refractive index of running buffer and sample solution

35 Reference surfaces If sample matrix and running buffer differ, the bulk contribution can be subtracted by using a reference surface Should be placed upstream of the active surface Reference subtraction is important for kinetic and affinity analyses but is not necessary for concentration analysis

36 Referencing to Eliminate Bulk Effects Response RU Active surface Reference surface Corrected data 500 s

37 Controls, Controls, Controls Is there nonspecific binding? if YES, to what?» use a control surface try to control with alterations to sample and running buffer» ph, ionic strength, detergent Other chips CM3 CM4 Stoichiometry (% Ligand Activity) R max = (MW A /MW L ) * R L * S m

38 Design of Reference surfaces Unmodified surface» For low immobilization levels on the active surface» To check for non-specific binding to the dextran matrix Activated-deactivated surface» Treating the surface with the immobilization procedure, but omitting the ligand» Decreases the negative charge on the surface and may reduce nonspecific binding Surface immobilized with dummy ligand» A protein that does not bind the analyte may be immobilized to approx. the Active Ligand same Surface level as the ligand to mimic the active Control surface Surface as closely as possible

39 Summary of the interaction Set up See the baseline stability. Sensorgram Ligand immobilization Ligand is immobilized onto the surface. Interaction Injection of analyte

40 General Steps of a Biacore Assay Surface preparation Sample injection Regeneration Evaluation

41 Regeneration Remove bound analyte completely from the surface The activity of the surface must remain unaffected Efficient regeneration is crucial for high-quality data

42 Testing regeneration conditions Response good Cycle 1 Cycle 2 Efficient regeneration removes all bound analyte bad Time A second injection of analyte reveals whether the ligand is still fully active Repeated cycles of analyte and regeneration injections is required to elucidate the condition

43 Optimization of Regeneration C P o o R U Response T i s Selection of regeneration» Contact time and flow» Harshness Impact on ligand activity» Reproducibility» Baseline Stability

44 Regeneration Acidic Basic Hydrophobic Ionic Strong ph < 2 glycine/hcl Formic Acid H 3 PO 4 Intermediate ph < glycine/hcl Formic Acid H 3 PO 4 Weak ph > 2.5 glycine/hcl Formic Acid H 3 PO 4 ph > 10 NaOH KOH ph 9-10 glycine/naoh NaOH KOH ph < 9 HEPES/ NaOH 50% ethylene glycol 40% ethylene glycol 25% ethylene glycol 6M guanidine chloride 2M MgCl 2 4M KCl 3M KSCN 1M NaCl

45 Interpreting trends in analyte binding & baseline responses (1) General guidance» Ideal regeneration: Analyte response is constant after repeated injections and within 10% of the level in the 1 st injection» Too mild conditions: The analyte response decreases and the baseline response increases» Too harsh conditions: The analyte response decreases and the baseline response is constant or decreases

46 Interpreting trends in analyte binding & baseline responses (2) Analyte binding response

47 Interpreting trends in analyte binding & baseline responses (3) Baseline response

48 Summary Establish immobilization procedure» What to immobilize» How to immobilize» How much to immobilize Evaluate ligand binding capacity Establish appropriate control and reference surfaces Establish regeneration conditions Assess surface stability» Monitor baseline stability» Monitor binding capacity

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