SETUP
Insufficient Ligand Immobilization
What you see
- Low or no RU response after analyte is flowed
- Flat sensorgram with no binding signal
What to try
- Increase ligand concentration
- Increase coupling time
- Change surface chemistry
Guide
Decoding SPR Sensorgrams
A field guide to every sensorgram shape we've seen across hundreds of labs in 15+ countries — what it means, and how to fix it.
The 5 Phases of a Sensorgram (BASDR)
Reading the cards: what you see what you would expect injection the problem
B
Baseline
Buffer flows over the sensor, establishing a stable reference signal.
A
Association
Analyte moves to the surface via mass transport and binds to the ligand.
S
Steady-State
Net rate of bound analyte is zero. Saturation may or may not be reached.
D
Dissociation
Buffer returns; analyte dissociates from the ligand.
R
Regeneration
Low pH buffer removes remaining bound analyte to restore the surface.
No exact match. Try another symptom, or contact us: we have likely seen it.
Part 1
Setup & Ligand Issues
Problems with ligand immobilization, regeneration, and sensor chip preparation.
SETUP
Incomplete Surface Regeneration
What you see
- Analyte not fully cleared between cycles
- Progressive baseline increase across runs
- Reduced binding capacity in subsequent cycles
What to try
- Optimize regeneration buffer composition, pH, and ionic strength
- Follow with original running buffer to re-establish baseline
- Check for non-specific binding; increase flow rate and regeneration time
SETUP
Rebinding Effects
What you see
- Analyte rebinds to nearby ligands after dissociation
- Dissociation does not follow a single exponential
- Baseline never recovered in dissociation phase
What to try
- Decrease ligand density on sensor; increase flow rates
- Use single-cycle kinetics
- Inject soluble ligand as competitor during dissociation
SETUP
Noisy Data
What you see
- High noise obscuring binding signal
- Erratic RU fluctuations across the entire run
What to try
- Increase analyte concentration
- Increase amount of immobilized ligand on sensor
Part 2
Assay Issues
Issues arising from analyte preparation, buffer composition, and assay conditions.
ASSAY
Buffer Mismatch (Bulk Shift)
What you see
- Square-shaped artifact in the binding curve
- Large, rapid RU changes at start and end of injection
- Analyte and running buffer compositions differ
What to try
- Match analyte buffer components with running buffer
- Dialyze or filter analyte into the same running buffer
- Run buffer-only reference and subtract from analyte experiment
ASSAY
Carryover Between Cycles
What you see
- Baseline increases across subsequent cycles
- Sudden buffer jumps or spikes at the start of analyte injection
- Often caused by high salt or sticky analyte
What to try
- Verify complete regeneration between cycles
- Reformulate analyte buffer
- Add extra washing steps between injections
ASSAY
Analyte Aggregation
What you see
- Erratic, irregular or noisy binding curves
- Sharp transient spikes or jumps in signal
- Abnormally high RU and poor model fitting
What to try
- Check protein folding and monodispersity
- Reformulate analyte buffer; replenish with fresh soluble analyte
- Filter or centrifuge to remove aggregates before injection
ASSAY
Mass Transport Limitations (MTL)
What you see
- Slow, gradual RU increase during association — faster at higher flow rates
- Analyte diffusion slower than binding rate
- Poor data modeling at low analyte concentrations
What to try
- Use higher flow rates and low-viscosity buffer
- Decrease ligand density on the sensor
- Use MTL-corrected models for data fitting
P4PRO with AffiPump is designed to minimize MTLs by delivering analyte quickly and evenly across the sensor surface.
ASSAY
Non-Specific Binding (NSB)
What you see
- Steep RU increase from analyte interaction with sensor surface
- High signal in reference channel — reflects diffusion, not binding
- Poor model fitting
What to try
- Increase salt concentration; adjust buffer pH
- Add BSA or non-ionic surfactants (e.g., Tween 20)
- Change ligand orientation or sensor surface chemistry
Part 3
Operational, Instrumentation & Other Issues
Issues arising from instrument state, fluidics, temperature, and compound effects.
OPERATIONAL
Baseline Drift
What you see
- Gradual upward or downward RU drift during the run
- May indicate temperature fluctuations, unstable sensor, or contamination
What to try
- Stabilize temperature in the instrument environment
- Replace sensor; equilibrate system with sufficient buffer
- Flow running buffer until baseline is stable
OPERATIONAL
Air Bubble or Clog in Lines
What you see
- Sudden, unpredictable RU spikes and drops during injection and dissociation
- Erratic signal not correlated with binding events
What to try
- Check instrument for fluidic leaks; purge lines
- Degas samples before injection
- Perform scheduled instrument maintenance
OPERATIONAL
Injection Failure
What you see
- No RU response after analyte injection
- May be caused by valve malfunction, air in syringe, or software error
What to try
- Check injection valves and syringe for air
- Restart software and re-run system maintenance
INSTRUMENTATION
Rounded Peak
What you see
- Inefficient analyte dissociation from the ligand
- Over- or under-estimation of binding affinity
- Poor fit with standard 1:1 global model
What to try
- Implement a regeneration step
- Use a fit model other than 1:1 global fit
COMBINED
Combination Effects
What you see
- MTLs, protein aggregation, and buffer mismatch simultaneously present
- Linear upward climb during association with no equilibrium plateau
- Steady higher baseline; poor fitting across all models
What to try
- Diagnose each effect individually before applying fixes
- Address buffer mismatch first, then aggregation, then MTL
- Refer to Parts 1 & 2 for individual fix protocols