Binding Characterization

Binding Characterization: SPR and BLI

SPR vs BLI Decision Matrix

| Factor | Choose SPR | Choose BLI | |--------|------------|------------| | Sensitivity | Small molecules, fragments (10μM) | Fast dissociation in BLI dip | Increase analyte concentration | | Low expression** | Not enough signal | Increase biosensor loading |

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Mass transport considerations

Mass transport limitation occurs when analyte cannot diffuse to the surface fast enough to maintain equilibrium. This distorts kinetic parameters.

Symptoms

• Observed kon appears slower than true kon
• Linear association phase (instead of exponential)
• kon varies with ligand density
• Rmax varies with flow rate

When mass transport matters

• High-affinity interactions (kon >10^6 M^-1s^-1)
• High ligand density (>500 RU)
• Slow flow rates (9): Use slightly acidic buffer

Reference subtraction

Always include:

• Blank reference channel (no ligand)
• Buffer-only injections
• Non-specific binding controls

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Regeneration conditions

SPR regeneration scouting (try in order)

| Condition | Targets | Caution | |-----------|---------|---------| | 10 mM Glycine pH 2.0-2.5 | Most protein-protein | May denature ligand | | 10 mM Glycine pH 1.5 | Strong interactions | Harsh, limit exposure | | 1-2 M NaCl | Ionic interactions | Mild, try first | | 10 mM NaOH | Very stable ligands | Can hydrolyze proteins | | 10 mM Glycine pH 9-10 | Acid-stable proteins | Can aggregate | | 10 mM EDTA | His-tag, metal-dependent | Strips Ni-NTA | | 4 M MgCl2 | Hydrophobic interactions | Check ligand stability |

Regeneration protocol

1. Start with mildest condition (high salt)
2. Test 30s contact time
3. Verify complete dissociation (return to baseline)
4. Verify retained ligand activity (repeat binding)
5. Use shortest effective contact time

BLI tips

• Tips are often disposable (no regeneration needed)
• For reuse: Same conditions as SPR, but shorter exposure
• Anti-His tips: 10 mM Glycine pH 1.5, 30s
• Streptavidin tips: Generally not regenerable

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Common artifacts and solutions

Biphasic binding

Symptoms: Two-rate association or dissociation Causes:

• Sample heterogeneity (aggregates)
• Ligand heterogeneity (multiple conformations)
• Avidity effects (bivalent analyte)

Solutions:

• Filter/centrifuge sample
• Use monovalent Fab fragments
• Reduce ligand density
• Fit to heterogeneous model

Negative dissociation

Symptoms: Signal increases during dissociation phase Causes:

• Ligand leaching from surface
• Analyte aggregation on surface
• Reference channel drift

Solutions:

• Use capture antibody instead of direct immobilization
• Increase buffer stringency
• Better reference subtraction

Hook effect

Symptoms: Signal decreases at high analyte concentrations Causes:

• Surface saturation + rebinding suppression
• Crowding effects

Solutions:

• Reduce analyte concentration range
• Reduce ligand density
• Use smaller analyte fragments

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Kinetic data quality checklist

Before analysis

• [ ] Reference-subtracted properly
• [ ] Buffer injection shows flat baseline
• [ ] Rmax consistent across concentrations
• [ ] No systematic drift during association
• [ ] Complete regeneration (return to baseline)
• [ ] Duplicate/triplicate injections consistent

Fitting quality

• [ ] Residuals randomly distributed (no systematic deviation)
• [ ] Chi² 10^7 M^-1s^-1), where rates are unreliable
• koff too fast to sample (> 0.1 s^-1) or too slow to measure in the dissociation window (> theoretical maximum (aggregation or avidity)
• Large difference between kinetic and equilibrium KD

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References

Platform comparisons

• BLI vs SPR Comparison - Sartorius
• BLI vs SPR - Nicoya

SPR protocols

• SPR Guidelines - van der Merwe, Oxford
• SPR Experiment Guide - Duke DHVI

Troubleshooting

• 4 Ways to Reduce NSB in SPR - Nicoya
• 3 Ways to Limit Mass Transfer Effects - Nicoya
• Suppressing NSB in BLI - ACS Omega

Regeneration

• SPR Regeneration - SPRpages
• Mastering Regeneration - Nicoya

Mass transport

• Mass Transport Limitation in SPR - PMC
• Mass-Transfer Kinetics - SPRpages

Source & license

This open-source skill is cataloged on AgentStack and links to its original source — we do not rehost the code.

• Author: adaptyvbio
• Source: adaptyvbio/protein-design-skills
• License: MIT

Install and usage instructions live in the source repository linked above.