Overview
This protocol walks through the complete Starter Test Kit experiment: EDC/NHS covalent immobilization of Anti-IgG on a carboxyl sensor, followed by a 5-concentration IgG ligand binding assay with KD determination. It is the recommended first experiment for new P4SPR 2.0 users.
Estimated Time
3–4 h
Sensor
Carboxyl
Running Buffer
PBS 1X pH 7.4
Injection Volume
250 µL
General Guidelines
Before you begin
- Manual injection: Inject at approximately 50 µL/s. Using a 1 mL syringe, 1 drop ≈ 50 µL.
- Injection volume: Minimum 200 µL per channel; 250 µL recommended to avoid air bubbles.
- Removing air pockets: Inject briefly at 100 µL/s or perform pulsed injections. Alternatively, inject 200 µL of detergent solution (0.5% SDS or 1% Tween 20) and rinse thoroughly with 10× volume H2O.
- Baseline stabilization: Inject running buffer and hold for at least 5 minutes contact time.
Materials
Kit Contents
| Component | Description | Qty | Storage |
|---|---|---|---|
| EDC aliquot | N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide HCl — ready to use | 250 µL | −18°C |
| NHS aliquot | N-Hydroxysuccinimide — ready to use | 250 µL | −18°C |
| Blocking solution | 1 M Ethanolamine pH 8.5 | 10.5 mL | 2–8°C |
| Immobilization buffer | Sodium acetate 10 mM, pH 5.0 | 10.5 mL | 2–8°C |
| Regeneration solution | Glycine-HCl 10 mM pH 2.5 | 10 mL | 2–8°C |
Warning: Thaw EDC and NHS aliquots immediately before use. Mix together just prior to injection — do not pre-mix and store.
Additional Materials Required
Consumables
- 50 mL filtered DI water (0.22 µm)
- 1 mL syringes
User-Supplied Reagents
- Anti-IgG protein (capture molecule)
- IgG (ligand / analyte)
- PBS 1X pH 7.4 (running buffer — if not supplied in kit, filter through 0.22 µm)
Solution Preparation
Protein (Anti-IgG) Solution
- Dilute Anti-IgG to 10 µg/mL (0.01 mg/mL) in immobilization buffer (sodium acetate 10 mM pH 5.0, supplied).
- Keep on ice until use. Allow to reach room temperature before injection.
Ligand (IgG) Solutions — 3× Serial Dilution from 72 nM
Prepare 5 concentrations in running buffer (PBS). Start at 72 nM and serial dilute 3×:
| # | Concentration | Dilution | Volume |
|---|---|---|---|
| 1 | 0.9 nM | Lowest | 250 µL |
| 2 | 2.7 nM | 250 µL | |
| 3 | 8 nM | 250 µL | |
| 4 | 24 nM | 250 µL | |
| 5 | 72 nM | Highest | 250 µL |
Protocol
Setup
- Install the carboxyl sensor (gloved hands, by edges only). Lower and lock the arm.
- Launch AffiLabs.core and click the Power button (⏻) in the Transport Bar. 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. Load the built-in Amine method template from the Template Gallery. The IM cycle is preset to 30 minutes to cover activation, protein immobilization, and blocking. Add 5 Binding cycles for the IgG concentrations. Click Save Method, then Add to Queue.
- Once solutions are ready, click Start Cycle Record.
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 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 Anti-IgG solution (0.01 mg/mL in 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 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 (5 Concentrations)
- Inject 250 µL of the lowest IgG concentration (0.9 nM) 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.
- Inject 250 µL 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: 2.7, 8, 24, and 72 nM (lowest → highest).
- After the final baseline cycle, click Stop (⏹).
Shutdown
- Inject abundant filtered DI water to rinse all used channels.
- Follow the shutdown procedure (User Guide, Chapter 8).
- Remove sensor (User Guide, Chapter 9).
Data Analysis
Load & Measure
- Edits tab → Load Data → select auto-saved .xlsx, or double-click the experiment in the Notes tab.
- For each Binding cycle: place the green cursor at injection start and the red cursor at the end of contact period. ΔSPR auto-saves.
- Set the reference channel. Use Lock cursors to step through all cycles at the same timepoint.
KD Fitting
- In the Binding subtab, select Langmuir 1:1 model.
- Click Calculate KD.
- Outputs: KD, Rmax, R².
Expected result (Anti-IgG / IgG model system): KD ≈ 14.9 ± 0.1 nM, Rmax ≈ 349 ± 1 RU, R² = 1.0000. Your values may differ slightly depending on sensor batch and preparation.
Related Protocols
- Protocol #1 — Covalent Immobilization of Proteins (EDC/NHS) — general-purpose version without kit reagents
- Protocol #4 — NaCl Calibration — verify sensor sensitivity before running assays
- Protocol #2 — Metal Chelation (Ni-NTA / His-Tag)
- Protocol #3 — Streptavidin Capture
PR-05 — Affinité Instruments Protocol Series · V4 (updated for AffiLabs.core) · April 2026