Apr 15, 2025
Creoptix Protein Immobilization Protocol: Biotin Capture on Streptavidin Chip
- Eda Capkin1,2,3
- 1Diamond Light Source;
- 2Research Complex at Harwell;
- 3ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Eda Capkin 2025. Creoptix Protein Immobilization Protocol: Biotin Capture on Streptavidin Chip. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6dym1vqe/v1
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: February 04, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 119548
Keywords: Immobilized Proteins, Surface Capture Strategy, Grating coupled interferometry, Creoptix, Biotin tagged protein, Streptavidin coated surface
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abstract
This protocol provides a quick yet comprehensive method for the capture of various biotinylated
proteins on a PCH-STA chip, that preserves protein activity. The procedure covers system preparation, chip conditioning, protein capture, and subsequent analysis using the Creoptix WAVEsystem. It is a methods of surface modification that includes surface priming with the running buffer, protein capture, and final wash steps. The protein capture step requires optimization of the target protein concentration and injection duration to achieve desired response levels.
Materials
PCH-STA: PCH-STA Pre-immobilized streptavidin, thick PC hydrogelMalvern PanalyticalCatalog #9060010
Borate buffer: 0.1 M sodium borate, 1 M NaCl pH 9.0XanTec bioanalytics GmbHCatalog #B BELU-50ML
10x HBS-P: HBS-P+ Buffer 10×CytivaCatalog #BR100671
Safety warnings
Do not set the Flow Cell Temperature below 10 and/or 15°C. At lower temperatures, condensation occurs on the chip.
Before start
The composition of the running buffer used will differ for each protein. To ensure the best chances of success, conduct a thorough literature search for information on you target protein’s buffer stability and any relevant experimental parameters from published immobilization assays.
Instructions:
Instructions:
The running buffer will differ for each protein. We highly recomment that a literature review should proceed any study to gather background information on protein stability and relevant experimental parameters from other immobilization assay used previously.
Variables that are for the immobilization steps indicated the table below.
| A | B | |
| Running buffer composition | [X] | |
| Protein concentration (μg/mL) | [Y] | |
| Protein injection duration (s) | [Z] |
Variables definitions
Setting up WAVEsystem
Setting up WAVEsystem
20m
20m
Prepare 200 mL running buffer by diluting stock solution (normally 10x) with Miili-Q water.
20m
Degas the buffer for 00:02:00 in an ultrasonic bath.
2m
Keep 40 mL running buffer aside for preparation of samples and other solutions.
Insert the tubes into the remainder of the running buffer and insert the PCH-STA chip into the instrument.
Select "new buffer and new chip" in the pop-up window. This will prime the system and perform the chip alignment.
During the chip alignment, check the profile and the amplitude levels at the end of the aligment step.
Example amplitude profile during the chip alignment.
Note
Manual checks to determine if the chip is functioning properly:
1) The amplitudes for all channels should be above 50 (a.u.)
2) During chip alignment the all amplitude profiles shows triangular spikes which return to flat baselines at the end of the alignment
PCH-STA chip conditioning
PCH-STA chip conditioning
In the Device tab, start Priming and Chip Prime. Repeat at least 2x.
Screenshot of Creoptix software to perform Prime and Chip prime
To condition the chip go to Conditioning on the Measure tab.
Screenshot of Creoptix software to perform Conditioning
Select chip type as PCH-STA chip.
Define the running buffer.
Create series and prepare samples as indicated in the series.
| 1 | 2 | 3 | 4 | 5 | |
| A | Borate buffer | Running buffer | |||
| B | |||||
| C | |||||
| D | |||||
| E | |||||
| F |
| 6 | 7 | 8 | |
| A | |||
| B | |||
| C | |||
| D | |||
| E | |||
| F |
Go to the series and run the measurement.
| Step | Solution | Concentration | Injection duration | Flow rate (ul/min) | |
| Conditioning | Borate buffer | 0.1M borate, 1M NaCl pH 9.0 | 180 | 10 | |
| Wash (3x times) | Running buffer | 1X buffer | 60 | 100 |
Different steps in conditioning series.
Check the response and overall profile for the conditioning step.
Example profile for the chip conditioning step.
Note
Manual checks to determine if the chip is working:
1) The first injection with borate buffer gives a response between 3 000 - 4 000 pg/mm2
2) During the wash steps with running buffer the baseline should return to almost 0 pg/mm2 and be mostly flat.
Biotin (AVI) Tagged protein capture
Biotin (AVI) Tagged protein capture
Start short injection duration (i.e. 60 s) at low protein concentration to check the capture levels. Depending on the protein capture response, the concentration and injection duration can be increased.
In the Measure tab, choose Immobilization for the protein capture.
Screenshot of Creoptix software to perform immobilization
Select the type of immobilization as Capturing biotinylated ligand to streptavidin surface
Set the flow path to the appropriate flow channel(s) protein capture step e.g. FC2, FC3, or FC4.
FC = flow channel
Enter the details of the protein, concentration, and injection time in to the software.
The immobilization target level of the protein can also be added if known, but this is not required.
Click Create Series.
Go to Autosampler section in the method. Check all the solutions and their locations in the specified vial or plate.
Calculate the required immobilization level based on the equation given the below.
Rmax=(MW analyte/MW ligand) x Immobilized Ligand(pg/mm2) x Stochiometry
Immobilization level should be around less than 70 Rmax.
Table 2. Biotin tagged protein capture method summary on the PCH-STA chip surface to check the protein capture level.
| A | B | C | D | E | |
| Wash (2x times) | Running buffer | 60 | 10 | ||
| Capture | Biotin tagged protein | 10 μg/mL | 60 | 10 | |
| Wash | Running buffer | 60 | 10 |
A: Step; B:Solutions; C: Concentrations; D:Injection Duration (s); E:Flow rate (uL/min)
Dilute the protein in the running buffer to the desired immobilization concentration. As a start, 10 µg/µL protein solution can be tested to check capture response level.
Place the samples according to the series in autosam pler.
Go to series in this method and start the capture step.
Check the capture protein response from the Measurements section in the project tree based on theoretical Rmax calculation
Protocol references
1. Kartal Ö, Andres F, Lai MP, Nehme R, Cottier K. waveRAPID—A Robust Assay for High-Throughput Kinetic Screens with the Creoptix WAVEsystem. SLAS DISCOVERY: Advancing the Science of Drug Discovery. 2021;26(8):995-1003. doi:10.1177/24725552211013827.