Summary 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.
Binding Characterization: SPR and BLI
SPR vs BLI Decision Matrix
Factor Choose SPR Choose BLI Sensitivity Small molecules, fragments (<500 Da) Large complexes, antibodies Throughput Low-medium (serial) High (96-well parallel) Sample purity Required (clogs fluidics) Tolerates crude lysates Kinetic resolution Higher (better for fast kinetics) Lower Mass transport More sensitive (may distort kon) Less sensitive Maintenance High (fluidics system) Low (dip-and-read) Sample consumption Higher (continuous flow) Lower Cost per experiment Lower chip cost, higher run cost Higher tip cost, lower run cost
Key differences
SPR (Surface Plasmon Resonance)
Mechanism : Detects refractive index changes at gold surfaceSurface : Gold chip with dextran matrix (CM5, CM7, etc.)Flow : Continuous microfluidicsBest for : Small molecules, high-affinity, precise kon/koff
BLI (Biolayer Interferometry)
Mechanism : Measures optical interference pattern shift
Surface : Fiber optic biosensor tips (SA, Ni-NTA, AHC)
Flow : Dip-and-read (no microfluidics)
Best for : High-throughput, crude samples, antibody screening
Troubleshooting: Why BLI works but SPR doesn't Cause Mechanism Solution Hydrophobic CDRs Adsorb to SPR gold/dextran surface Add 0.05% Tween-20, use CM7 chip with longer dextran Aggregation Mass transport artifacts in SPR fluidics Filter sample (0.22μm), reduce ligand density High instability Degrades during continuous flow Shorter cycle time, add stabilizers (trehalose 5%) Charge mismatch Nonspecific binding to charged dextran Adjust buffer pH ±1 from pI, add BSA 1mg/mL Slow dissociation Long regeneration needed (damages ligand) Use BLI (disposable tips)
Why SPR works but BLI doesn't Cause Mechanism Solution Small analyte BLI less sensitive for <10 kDa Use SPR with appropriate chip Weak affinity (KD >10μM) Fast dissociation in BLI dip Increase analyte concentration Low expression Not enough signal Increase biosensor loading
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 (<30 μL/min in SPR)Large analytes (slow diffusion)
Mitigation strategies Strategy SPR BLI Reduce ligand density <200 RU for high-affinity <0.5 nm shift loading Increase flow rate 50-100 μL/min Increase shake speed (1000 rpm) Use oriented immobilization His-tag capture Biotinylated ligand Include in fitting Mass transport model (kt) Usually less critical
Nonspecific binding mitigation
Buffer additives (ranked by effectiveness) Additive Concentration Mechanism Best For BSA 0.5-1 mg/mL Blocks hydrophobic sites General use Tween-20 0.02-0.05% Prevents surface adsorption Hydrophobic analytes Trehalose 1-5% Stabilizes + blocks Unstable proteins Sucrose 5% BLI-specific blocker BLI tips Carboxymethyl dextran 1 mg/mL Competitive blocking SPR with charged proteins NaCl 150-500 mM Reduces ionic interactions Charged proteins
pH optimization
Keep buffer pH at least 1 unit away from analyte pI
pI near 7: Use pH 6.0 or 8.0 buffer
Acidic proteins (pI <5): Use neutral or basic buffer
Basic proteins (pI >9): Use slightly acidic buffer
Reference subtraction
Blank reference channel (no ligand)
Buffer-only injections
Non-specific binding controls
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
Start with mildest condition (high salt)
Test 30s contact time
Verify complete dissociation (return to baseline)
Verify retained ligand activity (repeat binding)
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
Common artifacts and solutions
Biphasic binding Symptoms : Two-rate association or dissociation
Causes :
Sample heterogeneity (aggregates)
Ligand heterogeneity (multiple conformations)
Avidity effects (bivalent analyte)
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
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
Reduce analyte concentration range
Reduce ligand density
Use smaller analyte fragments
Kinetic data quality checklist
Before analysis
Fitting quality
Red flags
kon approaching mass transport limit (>10^7 M^-1s^-1)
koff faster than data acquisition (< 0.01 s^-1 requires faster sampling)
Rmax >> theoretical maximum (aggregation or avidity)
Large difference between kinetic and equilibrium KD
References
Platform comparisons
SPR protocols
Troubleshooting
Regeneration
Mass transport 