Overview
This protocol uses streptavidin as a capture layer on biotin-coated gold sensor chips. Streptavidin binds irreversibly to the biotin surface, creating a high-affinity platform for subsequent protein immobilization and ligand binding assays. This two-layer approach (biotin → streptavidin → protein) provides a versatile, oriented capture surface without the need for chemical activation.
Materials
Sensor Chips
- Au sensors + biotin (available from Affinité)
Reagents
- Streptavidin (Millipore Sigma SA101 or equivalent)
- Ethanol 95% (EtOH)
Buffers & Samples
- Distilled water (DI)
- Running buffer: PBS 1X, pH 7.4 (Corning Cellgro 21-040-CV) or equivalent
- Protein solution in running buffer
- Ligand solutions in running buffer
Solution Preparation
Prepare all solutions before starting and ensure they are at room temperature before injection.
| Solution | Preparation |
|---|---|
| Streptavidin | 0.025 mg/mL in DI water (1 mL) |
| Protein solution | 0.02 mg/mL in running buffer |
| Ligand solutions | Serial dilutions in running buffer (300 µL each) |
| Running buffer (PBS) | PBS 1X, pH 7.4 — used for washes and ligand dilutions |
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 (~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: After immobilization, streptavidin chips should be used immediately. Do not store overnight — the captured protein layer degrades.
Protocol
Setup
- Insert the biotin-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, 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 streptavidin capture + 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.
Streptavidin Capture
- Inject 250 µL of running buffer (PBS) in all channels. Hold for 5 minutes contact time to establish a stable baseline.
- Inject 250 µL of streptavidin (0.025 mg/mL in DI water) into each channel. Hold for 5 minutes contact time.
- Wash all channels with 250 µL DI water. Hold for 2 minutes contact time.
- Inject 250 µL running buffer (PBS). Hold for 2 minutes contact time or until the signal stabilizes.
Protein Immobilization
- Inject 250 µL of protein solution (e.g., antibody, 0.01 mg/mL in running buffer) 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.
Binding Assay
- Inject 250 µL of the least concentrated ligand 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 steps 1–2 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.
System Wash
- 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).
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 (Kd ~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
- Protocol #1 — Covalent Immobilization of Proteins (EDC/NHS)
- Protocol #2 — Metal Chelation Immobilization of His-Tagged Proteins
PR-03 — Affinité Instruments Protocol Series · V4 (updated for AffiLabs.core) · April 2026