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binding-characterization

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UpdatedMarch 4, 2026 at 07:22

Guidance for SPR and BLI binding characterization experiments. Use when: (1) Planning binding kinetics experiments, (2) Troubleshooting poor/no binding signal, (3) Interpreting kinetic data artifacts, (4) Choosing between SPR vs BLI platforms.

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BioTender-max

BioTender-max/ProteinClaw

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SKILL.md

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name	binding-characterization
description	Guidance for SPR and BLI binding characterization experiments. Use when: (1) Planning binding kinetics experiments, (2) Troubleshooting poor/no binding signal, (3) Interpreting kinetic data artifacts, (4) Choosing between SPR vs BLI platforms.
license	MIT
category	experimental
tags	["binding","spr","bli","validation"]
source	https://github.com/adaptyvbio/protein-design-skills

Binding Characterization: SPR and BLI

SPR vs BLI Decision Matrix

Factor	Choose SPR	Choose BLI
Sensitivity	Small molecules (<500 Da)	Large complexes, antibodies
Throughput	Low-medium (serial)	High (96-well parallel)
Sample purity	Required (clogs fluidics)	Tolerates crude lysates
Kinetic resolution	Higher (fast kinetics)	Lower
Mass transport	More sensitive	Less sensitive
Maintenance	High (fluidics)	Low (dip-and-read)

Troubleshooting Matrix

Problem	Mechanism	Solution
Hydrophobic CDRs adsorb to SPR surface	Gold/dextran surface	Add 0.05% Tween-20, use CM7 chip
Aggregation in SPR fluidics	Mass transport artifacts	Filter sample (0.22μm), reduce ligand density
Protein degrades during continuous flow	High instability	Shorter cycle time, add trehalose 5%
Small analyte (<10 kDa) on BLI	Low signal	Use SPR instead
Weak affinity (KD >10μM) on BLI	Fast dissociation	Increase analyte concentration

Mass Transport Considerations

Symptoms of mass transport limitation

Linear association phase (not exponential)
kon varies with ligand density
Rmax varies with flow rate

Mitigation

SPR: Reduce ligand density (<200 RU), increase flow rate (50-100 μL/min)
BLI: Reduce loading (<0.5 nm shift), increase shake speed (1000 rpm)

Regeneration Conditions (SPR)

Condition	Targets	Caution
10 mM Glycine pH 2.0-2.5	Most protein-protein	May denature ligand
1-2 M NaCl	Ionic interactions	Mild, try first
10 mM NaOH	Very stable ligands	Can hydrolyze proteins
10 mM EDTA	His-tag, metal-dependent	Strips Ni-NTA

Kinetic Data Quality Checklist

Reference-subtracted properly
Buffer injection shows flat baseline
Chi² < 10% of Rmax
kon and koff errors < 20%
KD from kinetics matches equilibrium KD (within 3-fold)

Red Flags

kon approaching mass transport limit (>10⁷ M⁻¹s⁻¹)
Rmax >> theoretical maximum (aggregation or avidity)
Large difference between kinetic and equilibrium KD

References

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name	binding-characterization
description	Guidance for SPR and BLI binding characterization experiments. Use when: (1) Planning binding kinetics experiments, (2) Troubleshooting poor/no binding signal, (3) Interpreting kinetic data artifacts, (4) Choosing between SPR vs BLI platforms.
license	MIT
category	experimental
tags	["binding","spr","bli","validation"]
source	https://github.com/adaptyvbio/protein-design-skills

Binding Characterization: SPR and BLI

SPR vs BLI Decision Matrix

Factor	Choose SPR	Choose BLI
Sensitivity	Small molecules (<500 Da)	Large complexes, antibodies
Throughput	Low-medium (serial)	High (96-well parallel)
Sample purity	Required (clogs fluidics)	Tolerates crude lysates
Kinetic resolution	Higher (fast kinetics)	Lower
Mass transport	More sensitive	Less sensitive
Maintenance	High (fluidics)	Low (dip-and-read)

Troubleshooting Matrix

Problem	Mechanism	Solution
Hydrophobic CDRs adsorb to SPR surface	Gold/dextran surface	Add 0.05% Tween-20, use CM7 chip
Aggregation in SPR fluidics	Mass transport artifacts	Filter sample (0.22μm), reduce ligand density
Protein degrades during continuous flow	High instability	Shorter cycle time, add trehalose 5%
Small analyte (<10 kDa) on BLI	Low signal	Use SPR instead
Weak affinity (KD >10μM) on BLI	Fast dissociation	Increase analyte concentration

Mass Transport Considerations

Symptoms of mass transport limitation

Linear association phase (not exponential)
kon varies with ligand density
Rmax varies with flow rate

Mitigation

SPR: Reduce ligand density (<200 RU), increase flow rate (50-100 μL/min)
BLI: Reduce loading (<0.5 nm shift), increase shake speed (1000 rpm)

Regeneration Conditions (SPR)

Condition	Targets	Caution
10 mM Glycine pH 2.0-2.5	Most protein-protein	May denature ligand
1-2 M NaCl	Ionic interactions	Mild, try first
10 mM NaOH	Very stable ligands	Can hydrolyze proteins
10 mM EDTA	His-tag, metal-dependent	Strips Ni-NTA

Kinetic Data Quality Checklist

Reference-subtracted properly
Buffer injection shows flat baseline
Chi² < 10% of Rmax
kon and koff errors < 20%
KD from kinetics matches equilibrium KD (within 3-fold)

Red Flags

kon approaching mass transport limit (>10⁷ M⁻¹s⁻¹)
Rmax >> theoretical maximum (aggregation or avidity)
Large difference between kinetic and equilibrium KD