binding-characterization

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

• SPR Guidelines - van der Merwe, Oxford
• BLI vs SPR - Nicoya