Covalent Immobilization of Proteins

Overview

This protocol describes the covalent immobilization of proteins (e.g., antibodies, peptides) on self-assembled monolayer (SAM) coated gold sensor chips using EDC/NHS coupling chemistry, followed by a ligand binding assay. It is the standard starting protocol for most P4SPR experiments involving protein targets.

Expected sensorgram profile for a covalent immobilization experiment. The SPR shift increases during protein immobilization and remains stable after blocking. Successive ligand injections produce step-wise binding responses.

Materials

Sensor Chips

Au sensors + 16-MHA (SAM01-5), or
Au sensors + AffiCoat (SAM02-5)

Chemicals

EDC — N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (Sigma-Aldrich E6383)
NHS — N-Hydroxysuccinimide, 98% (Aldrich 130672)
Ethanolamine hydrochloride, ≥99% (Sigma-Aldrich E6133)
Hydrochloric acid (HCl)
Sodium hydroxide (NaOH)
Sodium acetate

Buffers & Solutions

Distilled water (DI)
Running buffer: PBS 1X, pH 7.4 (Corning Cellgro 21-040-CV) or equivalent
Protein solution in immobilization buffer
Ligand solutions in running buffer

Solution Preparation

Prepare all solutions before starting the experiment. Ensure all solutions are at room temperature before injection.

Solution	Preparation
Immobilization buffer	Sodium acetate 10 mM, pH 5.0 (supplied in Starter Kit, or prepare and adjust with HCl)
Blocking solution	1 M ethanolamine, pH 8.5 (supplied in Starter Kit, or prepare and adjust with 0.1 M NaOH)
Protein solution	0.01 mg/mL (10 µg/mL) protein in immobilization buffer. Keep on ice until use; allow to reach room temperature before injection.
EDC/NHS activation mix	Thaw EDC and NHS aliquots (supplied in Starter Kit at −18°C). Mix together immediately before injection — do not pre-mix and store.
Regeneration solution	Glycine-HCl 10 mM, pH 2.5 (supplied in Starter Kit) — optional, for multi-cycle experiments
Ligand solutions	Serial dilutions in running buffer (250 µL each). Prepare 5 concentrations (e.g., 3× serial dilution from 72 nM).

Tip: Prepare larger volumes of EDC and NHS stock solutions, aliquot, and freeze at −20°C. Thaw and mix immediately before use to prevent degradation.

General Notices

Before you begin

Manual injection: Inject at approximately 50 µL/s. Using a 1 mL syringe, 1 drop ≈ 50 µL.
Removing bubbles: Inject at a higher flow rate (~200 µL/s) or use pulsed injections.
Injection volume: Minimum 200 µL per channel; 250 µL recommended to avoid air bubbles.
Baseline stabilization: Inject running buffer and hold for at least 5 minutes contact time before starting.

Critical: Always visually inspect channels for air bubbles before EDC/NHS activation, immobilization, and blocking steps. Air bubbles at these stages will compromise the surface.

Protocol

Setup

Insert the SAM-coated gold chip into the sample holder, lock in place, and inject 1 mL DI water in all channels.
Launch AffiLabs.core and click the Power button (⏻) in the Transport Bar. The button turns yellow while scanning for hardware, then calibration begins automatically (~30–60 s). When the QC dialog shows pass, click Continue — the power button turns green.
Select your user profile from the Lab Users panel.
In the Transport Bar, click Build Method. You have two options:
- Option A — Load the Amine template: Select the built-in Amine method from the Template Gallery. The IM cycle is preset to 30 minutes to cover activation, protein immobilization, and blocking. Binding cycles are added separately.
- Option B — Build from scratch: Add cycles manually (Baseline → IM → Binding × N). Set contact times per cycle.
Click Save Method, then Add to Queue.
Click Record (⏺) in the Transport Bar. The queue begins executing and a green “Recording” indicator appears. Confirm a stable baseline over 5 minutes. The baseline should not fluctuate more than 10 RU.

EDC/NHS Activation

Establish a stable baseline with running buffer (PBS) for 5 minutes contact time.
Thaw EDC and NHS aliquots. Mix together immediately before injection.
Inject 200 µL of fresh EDC/NHS mixture into both sample and control channels. Hold for 5 minutes contact time.
Wash all channels with 250 µL running buffer. Hold for 2 minutes contact time or until the signal stabilizes.

Protein Immobilization

Inject 250 µL of protein solution (0.01 mg/mL in immobilization buffer, sodium acetate pH 5.0) into the sample channel only. Hold for 5 minutes contact time.
Wash all channels with 250 µL running buffer. Hold for 2 minutes contact time or until the signal stabilizes.

Blocking

Inject 250 µL of 1 M ethanolamine pH 8.5 (blocking solution) into both channels. Hold for 5 minutes contact time.
Wash all channels with 250 µL running buffer. Hold for 2 minutes contact time or until the signal stabilizes.

Binding Assay

Inject 250 µL of the least concentrated ligand solution into both channels (sample and control). Hold for 5 minutes contact time.
Wash all channels with 250 µL running buffer. Hold for 2 minutes contact time or until the signal stabilizes.
Inject 250 µL of blocking solution and hold for 10 seconds, then re-inject 250 µL running buffer and hold for 2 minutes contact time or until stable.
Repeat steps 1–3 for each subsequent concentration (lowest to highest).
After the final baseline cycle, click Stop (⏹). Data auto-saves to Excel. Load in the Edits tab to measure ΔSPR per cycle using cursors.

Shutdown

Inject abundant filtered DI water to rinse all used channels.
Follow the shutdown procedure in AffiLabs.core (User Guide, Chapter 8).
Remove sensor (User Guide, Chapter 9).

Acceptance Criteria

After the immobilization and buffer wash, verify surface stability:

SPR shift check: (Running buffer 2 − Running buffer 1) / Running buffer 1 × 100 ≤ 10%

If the drift exceeds 10%, re-inject running buffer and allow additional stabilization time before proceeding to the binding assay.

Immobilization Methods Compared

Feature	PR-01 — Covalent (EDC/NHS)	PR-02 — Metal Chelation	PR-03 — Streptavidin Capture
Sensor chip	SAM01 (16-MHA) or SAM02 (AffiCoat)	SAM03 (Ni-NTA)	Biotin-coated Au
Surface prep	EDC/NHS activation (2 min)	NiCl₂ loading (5 min)	Streptavidin capture (20 min)
Blocking	Ethanolamine (10 min)	None	None
Bond type	Covalent (amide)	Non-covalent (coordination)	Non-covalent (K_d ~10⁻¹⁵ M)
Protein orientation	Random	Oriented (via His-tag)	Depends on capture strategy
Protein requirement	Any with primary amines	Must be His-tagged	Any
Overnight storage	Possible (refrigerated)	Possible (refrigerated)	Not recommended
Total hands-on time	~3–4 h	~30 min	~50 min

Related Protocols

PR-01 — Affinité Instruments Protocol Series · V4 (updated for AffiLabs.core) · April 2026