Sep 04, 2025
WaveRAPID - Protein Science course @ Aarhus University
This protocol is a draft, published without a DOI.
- Jan Nowak1
- 1Department of Molecular Biology and Genetics, Aarhus University, Universitetsbyen 81, 8000 Aarhus C, Denmark
Protocol Citation: Jan Nowak 2025. WaveRAPID - Protein Science course @ Aarhus University . protocols.io https://protocols.io/view/waverapid-protein-science-course-aarhus-university-g84tbzywp
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 01, 2025
Last Modified: September 04, 2025
Protocol Integer ID: 226163
Keywords: carbonic anhydrase, advantage of waverapid, short measurment time for each ligand, waverapid, ligand, furosemide, creoptix wavesystem, protein science course, refractive index change, drug discovery, sensor chip, resulting sensorgram, short pulse, local refractive index
Disclaimer
This protocol is heavily based on the "Training Guidebook: Protein – Small Molecule interaction with Creoptix WAVEsystem" published by Malvern. It is ment to be used for the Protein Science course at Aarhus University.
Abstract
The Creoptix WAVEsystem uses Grating-Coupled Interferometry (GCI) to measure label-free biomolecular
interactions in real time. The system detects refractive index changes upon
binding and dissociation, as light in the sensing arm undergoes a phase shift
depending on the local refractive index within the evanescent field.
In this experiment, carbonic anhydrase is immobilized on the sensor chip, and furosemide can be introduced
at varying concentrations using either the traditional multi-cycle mode or waveRAPID. In waveRAPID, short pulses from a single concentrated stock create a stepwise concentration increase, replacing the need for multiple separate injections. The resulting sensorgrams are analyzed to determine ka, kd, and KD. The advantage of waveRAPID is the short measurment time for each ligand, which makes it highly suitable for
screening efforts used in e.g. drug discovery.
Carbonic anhydrase is immobilized on a 4PCH chip beforehand, so your task it to perform a waveRAPID and determine the kinetics of binding.
Image Attribution
Creoptics kinetics guide
Materials
What you need for this experiment:
Consumables:
- Plastic snap vials (maximum volume 700 μL, dead volume 20 μL)
- Glass vials (maximum volume 1.8 mL, dead volume 400 μL)
- Snap cap lids
Reagents:
- 4PCH WAVEchip with CAII immobilized on the surface
- 1 mM Furosemide in 3% DMSO
- 1x PBS buffer + 3% DMSO
Troubleshooting
Safety warnings
Always double check that the autosampler layout fits the actual placement of the vials before initiating the experiment.
Before start
Make sure you have all reagents from Materials avalible.
WaveRAPID experiment
In WAVEcontrol software select RAPID kinetics from the Measure tab
Set Left Rack to 48 Vials Adapter with volume of 380 µL
Set Right Rack to 48 Vials Adapter with volume of 1400 µL
Select the Weak Binders pre-set on Affinity Range from the
Configuration panel, set flowpath to FC1 and FC2, and specify the running
buffer.
Double-click on empty well on autosampler and enter “Furosemide” as Designation and 100 µM as Concentration, as below:
Click Create Series
Dilute the furosemide 1 mM stock to 200 µL 100 μM in PBS 3% DMSO.
Prepare 1 mL DMSO Calibration sample by adding 0.5% of DMSO to the running buffer (PBS 3% DMSO).
Note
E.g., 5 μL of 100% DMSO + 995 μL of PBS 3% DMSO.
Apply reagents as displayed in the Autosampler view.
You can reposition the wells by dragging and dropping (e.g. Right-rack reagents together in Left
Rack).
Note
Remember to add the dead volume to the volumes displayed (20 µL for the 700 µl vials and 400 µl for the 1.5 ml glass vials).
Start series by selecting Run Measurement on Series tab.
WaveRAPID analysis
Select the series to be evaluated by clicking it in the project tree. In WAVEcontrol select Kinetic Analysis from the Evaluate & Simulate tab.
Adjustments and evaluations are done automatically and Evaluations tab will be opened.
The overall quality of the fitting is provided in Quality panel (right part). Model fitness can be also evaluated using the statistical analysis done automatically by Direct Kinetics analysis (an automated
evidence-based calculation of kinetic parameters with sound error) and shown in
the Summary Table (errors are in %)