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SPR Surface Chemistry
Explained

The sensor surface determines how your ligand attaches, whether it can be regenerated, and what sample quality you need.

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Why it matters

Two strategies, very different trade-offs

Every SPR experiment starts with the same decision: how do you get your ligand onto the sensor surface? The answer determines your surface density, ligand orientation, regeneration strategy, and the purity requirements for your sample.

There are two fundamentally different approaches — direct covalent immobilization (you chemically couple the protein to the surface) and capture-based immobilization (the sensor surface holds a capture agent that grabs your tagged protein). Each has clear advantages and specific limitations.

RANDOM ORIENTATION ✓ ? ✕ SENSOR SURFACE (Au) ligand analyte blocked

STRATEGY 01

Direct immobilization

Covalent · EDC/NHS amine coupling

✓No tag or modification required
✓Permanent — stable over many regeneration cycles (depends on the ligand)
!Requires purified protein (>80% homogeneous)
~Random orientation — some active sites may be buried
→Use Immobilization Scouting Kit to optimize preconcentration pH

Sensors

Carboxyl CMD AffiCoat

Kits

Amine Kit Amine CMD Kit Immo. Scouting
Step-by-step: Protocol #1 — Covalent (EDC/NHS) →
ORIENTATED CAPTURE ✓ ✓ ✓ CAPTURE LAYER SENSOR SURFACE (Au) capture agent ligand analyte

STRATEGY 02

Capture-based immobilization

Affinity · Tag or antibody interaction

✓Defined, orientated ligand presentation — all sites accessible
✓Most capture surfaces can be stripped and reloaded with fresh ligand
!Exception: streptavidin–biotin is effectively permanent — it can't be stripped
✓Most pre-coated sensors are ready to use — no coupling step
✓Works with less-pure samples (antibody-capture sensors)
!Requires a tag or modification (His, biotin, Strep, Fc…)

Sensors

Anti-His NTA Protein A Protein G Streptavidin Strep-Tactin XT Anti-AAVX

Kits

NTA Kit SA Kit IgG Capture Kits
Step-by-step: Protocol #2 — His-tag / NTA → Step-by-step: Protocol #3 — Streptavidin capture →

Side-by-side comparison

Direct immobilization
Capture-based
Tag required
None
His · Biotin · Strep · Fc…
Sample purity
>80% purified
Tolerates partially purified samples (antibody capture best)
Orientation
Random — some sites buried
Defined — all sites accessible
Surface lifetime
Permanent — very stable
Mostly renewable — strip and reload (not streptavidin)
Setup
EDC/NHS activation + coupling
Pre-coated sensors ready to use

Capture options at a glance

Capture surface
Tag on your protein
How strong
How to reset
Ni-NTA
His-tag
Moderate — ligand can slowly release (seen as baseline drift)
EDTA strips Ni²⁺ and ligand; recharge with 10 mM NiCl₂ · Protocol #2
Anti-His
His-tag
Stronger and more stable than NTA
Regenerate at low pH (glycine-HCl)
Protein A / Protein G
Fc region (IgG)
Strong, reversible
Regenerate at low pH (glycine-HCl)
Streptavidin
Biotin
Essentially irreversible (KD ~10⁻¹⁵ M) — very stable
Can't be stripped — regenerate the analyte only; new ligand = new sensor · Protocol #3
Strep-Tactin XT
Strep-tag II / Twin-Strep-tag
Very strong (low pM), yet reversible
3 M guanidine-HCl releases the captured protein; the Strep-Tactin XT layer stays on the chip (supplier reports 30+ cycles)

When to use each strategy

Direct immobilization

Choose covalent coupling when…

  • Your protein has no tag and can't be modified
  • You need a permanent, ultra-stable surface for long multi-day studies
  • Sample purity is high (>80%) and reproducible
  • You want to maximize surface density for low-MW analytes

Capture-based

Choose capture when…

  • Your protein carries a tag (His, biotin, Strep-tag, Fc region…)
  • You want consistent, orientated surface loading every run
  • Your sample is complex or partially purified
  • You need to regenerate the surface and reload fresh ligand (NTA, anti-His, Protein A/G)

Before you immobilize

How much ligand should I immobilize — and which partner goes on the surface?

Which partner goes on the surface? Immobilize the partner that best tolerates coupling and regeneration. If both work, put the larger one in solution: the SPR signal scales with the mass that binds, so a bigger analyte gives a bigger signal. Small molecules are the classic exception: they go in solution over a high-density protein surface, since they give only a small response.

  • Kinetics (ka, kd): use a low ligand density. A dense surface makes the association phase linear (mass-transport limited) and lets released analyte rebind during dissociation, which distorts the rate constants.
  • Small analytes or simple yes/no binding: use a higher density to maximize the signal.

Rmax = (MWanalyte / MWligand) × immobilized ligand level (RU) × stoichiometry

Worked example: a 150 kDa ligand immobilized at 1,000 RU and a 50 kDa analyte binding 1:1 give Rmax = (50 / 150) × 1,000 × 1 ≈ 333 RU. Stoichiometry is the number of analyte molecules each ligand can bind — an antibody has two binding sites, so for an antibody ligand it is 2. This is the theoretical maximum: the Rmax from your fit is usually lower, because not every immobilized ligand stays active — so comparing the two is a quick check of surface quality.

More on reading Rmax, density and kinetics: TN-03: The SPR sensorgram explained · Static vs. kinetic SPR · Blog: SPR sensorgram explained

Troubleshooting coupling and capture

Stuck mid-experiment? The most common problems — and what to check first.

Why do I get bubbles during EDC/NHS activation?

  • Check every channel for air bubbles before activation, immobilization and blocking — a bubble at any of these steps compromises the surface.
  • Inject at least 200 µL per channel; 250 µL is recommended to avoid trapping air.
  • To clear a bubble, inject at a higher flow rate (~200 µL/s) or use pulsed injections.
  • Degas buffers and let solutions reach room temperature before injecting — cold solutions release dissolved gas as they warm.
  • A stubborn bubble before the surface is activated: inject 200 µL of 0.5% SDS or 1% Tween 20, then rinse with 10× the volume of water.
  • Full procedure: Protocol #1 — Covalent immobilization.

Why is my immobilization level low?

  • Coupling buffer pH: the protein must carry a net positive charge to concentrate on the carboxyl surface, so use a pH below its pI. Protocol #1 uses 10 mM sodium acetate, pH 5.0; the Immobilization Scouting Kit finds the best pH for your protein.
  • No amine-containing buffers (Tris, glycine) in the protein solution — they react with the activated surface and compete with your protein.
  • Fresh EDC/NHS: aliquot and freeze the stocks, then thaw and mix immediately before injection — never pre-mix and store.

My protein coupled, but it barely binds its partner — why?

  • Amine coupling attaches the protein through its lysines at random. If lysines sit in or near the binding site, part of the surface is inactive.
  • Switch to a capture strategy (His-tag, biotin, Fc) for oriented presentation — see the table above.

Why does my baseline drift after His-tag capture?

  • His-tag binding to Ni-NTA is moderate, so captured ligand can slowly dissociate and the baseline drifts down. Anti-His capture holds more tightly.
  • Any surface needs time to settle: hold running buffer until the baseline varies less than 10 RU over 5 minutes before you start.
  • Baseline creeping up or drifting down between cycles? Check your regeneration: SPR regeneration guide →

How do I reduce non-specific binding?

  • Run a reference channel (same surface, no ligand) and subtract it.
  • Use AffiCoat, a zwitterionic surface that resists non-specific adsorption — suited to serum and cell lysates. How AffiCoat works →
  • Tween 20 in the running buffer (typically 0.005–0.05%) also helps.
Not working with a protein? TN-01 also covers DNA/RNA, lipids and membrane proteins (the long-chain alkanethiol SAM surface, which takes both hydrophilic and hydrophobic capture molecules) and complex matrices such as serum and cell lysate (the zwitterionic AffiCoat surface). Read TN-01 →

Go deeper

TN-01: Surface functionalization & coupling Every sensor surface, including DNA/RNA, lipids and membrane proteins. TN-01 as a PDF Download the technical note for the lab bench. AffiCoat for complex samples Reducing non-specific adsorption in serum and lysates.

Ready to choose your sensor?

Use the step-by-step selector on the Sensor Guide to get a recommendation based on your specific ligand tag and experiment type — then go straight to the catalog to build your order.

Sensor Selection Guide → Browse Catalog