Metal Chelation Immobilization of His-Tagged Proteins

Overview

This protocol describes the immobilization of histidine-tagged (His-tagged) proteins on Ni-NTA coated gold sensor chips via metal chelation, followed by an antibody binding assay. Unlike covalent EDC/NHS coupling (Protocol #1), metal chelation is non-covalent and orientation-specific — the His-tag anchors the protein in a defined orientation, leaving the binding site accessible. No activation or blocking steps are required, making this a simpler and faster protocol.

Ni-NTA metal chelation immobilization scheme. The Ni²⁺ ion on the NTA-coated gold surface coordinates with the His₆-tag on the protein, anchoring it in a defined orientation with the binding site exposed. Antibody (ligand) binds the accessible epitope. No activation or blocking chemistry is required.

Materials

Sensor Chips

Au sensors + Ni-NTA (SAM03-5)

Buffers & Solutions

Distilled water (DI)
Running buffer: PBS 1X, pH 7.4 (Corning Cellgro 21-040-CV) or equivalent
Nickel(II) chloride (NiCl₂) — for Ni²⁺ surface charging

Samples

His-tagged protein: 0.1 mg/mL in running buffer (1 mL)
Antibody (ligand) solutions: serial dilutions in running buffer (300 µL each)

Solution Preparation

This protocol requires minimal solution preparation compared to EDC/NHS coupling. Prepare all solutions before starting and ensure they are at room temperature before injection.

Solution	Preparation
NiCl₂ solution	10 mM NiCl₂ in DI water (500 µL per channel)
His-tagged protein	0.1 mg/mL in running buffer (PBS), 1 mL total
Antibody solutions	Serial dilutions in running buffer (300 µL each)
Running buffer (PBS)	PBS 1X, pH 7.4 — used for baseline, washes, and ligand dilutions

General Notices

Before you begin

Manual injection: Inject at approximately 50–100 µL/s. Using a 1 mL syringe, 1 drop ≈ 50 µL.
Removing bubbles: Inject at a higher flow rate (~100 µL/s) or use pulsed injections. Alternatively, inject 200 µL of 0.5% SDS or 1% Tween 20 and rinse thoroughly with 10× volume H₂O.
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 the immobilization step. Air bubbles at this stage will compromise the surface.

Protocol

Setup

Insert the Ni-NTA coated gold chip into the sample holder, lock in place, and flow DI water through all channels. Visually inspect to ensure there are no air bubbles.
Launch AffiLabs.core and click the Power button (⏻) in the Transport Bar. The button turns yellow while scanning, 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. Add cycles: Baseline → IM (for nickel loading + protein immobilization) → Binding × N. Set contact times per cycle. Click Save Method, then Add to Queue.
Click Record (⏺). The queue begins executing and a green “Recording” indicator appears. The baseline should not fluctuate more than 10 RU.

Nickel Loading

Inject 250 µL of running buffer (PBS) in all channels. Hold for 5 minutes contact time to acquire a stable baseline.
Inject 250 µL of 10 mM NiCl₂ in all channels. Hold for 5 minutes contact time to charge the NTA surface with Ni²⁺ ions.
Wash all channels with 250 µL running buffer. Hold for 2 minutes contact time or until the signal stabilizes.

Immobilization

Inject 250 µL of His-tagged protein (0.1 mg/mL in running buffer) into the sample channel only. Hold for 5 minutes contact time to allow binding to the Ni²⁺-charged NTA surface.
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 antibody 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.
Repeat with each subsequent antibody concentration (lowest to highest).
After the final injection, click Stop to end the run. Data is saved automatically to an Excel file (Raw_YYYYMMDD_HHMMSS.xlsx). Navigate to the Edit tab to review cycles and measure ΔSPR binding responses.

System Wash

Inject 20 mL of DI water to rinse all channels, then inject air to dry out the channels.

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-02 — Affinité Instruments Protocol Series · V4 (updated for AffiLabs.core) · April 2026