Feb 21, 2025
Creoptix Protein Immobilization Protocol: His-tag Capture via EDC/NHS chemistry
- 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: His-tag Capture via EDC/NHS chemistry. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2qynxl1y/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: August 02, 2024
Last Modified: February 21, 2025
Protocol Integer ID: 104519
Keywords: Immobilized Proteins, Surface Capture Strategy, His-capture, Carbodiimides/chemistry, Succinimides/chemistry , Grating coupled interferometry, Creoptix
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 outlines a comprehensive method for immobilizing His-tagged proteins on a PCH-NTA chip using the Creoptix WAVEsystem. The procedure involves chip conditioning, protein capture, and covalent immobilization through His capture and EDC/NHS chemistry. Key steps include surface activation with NiCl2, protein capture, and surface passivation with ethanolamine. It emphasizes the importance of optimizing protein concentration and injection duration to achieve desired immobilization levels. This method enables the stable attachment of His-tagged proteins in an oriented and active way for subsequent binding studies and is adaptable to various protein targets.
Materials
PCH-NTA: Pre-immobilized NTA Thick PC hydrogel Malvern PanalyticalCatalog #9060009
Borate buffer: 0.1 M sodium borate, 1 M NaCl pH 9.0XanTec bioanalytics GmbHCatalog #B BELU-50ML
EDTA: EDTA (0.5 M, pH 8.0, nuclease-free)Thermo Fisher ScientificCatalog #AM9260G
10x HBS-P: HBS-P+ Buffer 10×CytivaCatalog #BR100671
EDC: EDC*HClXanTec bioanalytics GmbHCatalog #C EDCHCL-1G
NHS:0.1 M NHS pH 5.0XanTec bioanalytics GmbHCatalog #B MN-50ML
NiCl2 :
1.option Nickel(II) chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #223387
2.optionNiCl2 loading bufferXanTec bioanalytics GmbHCatalog #B NILB-50ML
Ethanolamine:Ethanolamine quenching buffer:1 M Ethanolamine * HCl pH 8.5XanTec bioanalytics GmbHCatalog #B EA85-50ML
Safety warnings
Do not set the Flow Cell Temperature below 10 and/or 15°C. Lower temperature causes to the consensation in the chip.
Before start
The running buffer for each protein can be changed depending on the protein stability. Prior to start the method, a literature review should be done to check any background information on both protein stability and if any immobilization assay.
Instructions:
Instructions:
The running buffer for each protein can be changed depending on the protein stability. Prior to start the method, a literature review should be done to check any background information on both protein stability and if any immobilization assay.
Variables that are for the imobilization steps indicated the table.
| A | B | |
| Variable | Abbreviation for the variable | |
| Running buffer | [X] | |
| Protein concentration | [Y] | |
| Protein injection duration | [Z] |
This information will be different depending on the specific protocol and protein target.
Setting up WAVEsystem
Setting up WAVEsystem
20m
20m
Dilute 10X running buffer [X] to 1X reach a final volume of500 mL with Miili-Q water.
20m
Degas the buffer for 00:02:00 in a ultrassonic bath.
2m
Keep 40 mL from the 1X running buffer for preparation of samples and other solutions.
PCH-NTA chip conditioning
PCH-NTA chip conditioning
In the Device section, Start Priming and Chip Prime at least 2x.
Screenshot of Creoptix software to perform Prime and Chip prime
Conditioning of the chip is executed through wizard in Measure tab.
Screenshot of Creoptix software to perform Conditioning
Select chip type as PCH-NTA chip.
Define 1X running buffer.
Create series and prepare samples as indicated in the series.
| 1 | 2 | 3 | 4 | 5 | |
| A | Borate buffer | 0.25 M EDTA | Running buffer | ||
| B | |||||
| C | |||||
| D | |||||
| E | |||||
| F |
| 6 | 7 | 8 | |
| A | |||
| B | |||
| C | |||
| D | |||
| E | |||
| F |
Go to the series and run the measurement.
A: Step, B: Solution, C: Concentration, D: Injection duration, E: Flow rate (uL/min)
| A | B | C | D | E | |
| Conditioning | Borate buffer | 0.1M borate, 1M NaCl pH 9.0 | 180 | 10 | |
| Conditioning | EDTA | 250 mM | 180 | 10 | |
| Wash (3x times) | Running buffer | 1X buffer | 60 | 100 |
A: Step, B: Solution, C: Concentration, D: Injection duration, E: Flow rate (uL/min)
The graph after PCH-NTA chip conditioning
His Tagged protein immobilization
His Tagged protein immobilization
First, perform the capturing of His tagged ligands to Ni-NTA surface in the wizard (Table 2) to check whether the protein capture response at that concentration and duration is enough or not for the
immobilization step.
Perform Immobilization assaywithin the Measure Tab.
Screenshot of Creoptix software to perform immobilization
First select the type of immobilization as Capturing His Tagged ligands to the Ni-NTA surface
Select the Flow path as FC1 and FC2 for activation step.
Select FC2 for the capture step for His tagged protein and click create series. FC1 is used as reference “blank” channel. It should not have any protein in the chip.
Click Create Series.
Go to Autosampler section in the method. Check all the solution and their locations in the specified vial or plate.
Calculate the required immobilization level based on the equation given the below. Immobilization level should be around 70 to 100 Rmax.
Rmax=(MW analyte/MW ligand) x Immobilized Ligand(pg/mm2) x Stochiometry
Table 2. His tagged protein capture method summary on the Ni-NTA activated surface to check the protein capture level.
| A | B | C | D | E | |
| Wash (2x times) | Running buffer | 60 | 10 | ||
| Activation | NiCl2 | 500 uM | 120 | 10 | |
| Wash | Running buffer | 60 | 10 | ||
| Capture | His tagged protein | 20 ug/mL | 420 | 10 | |
| Wash | Running buffer | 60 | 10 | ||
| Regeneration | EDTA | 350 mM | 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 final immobilization concentration in 250 µL uL final volume. As a start, 15 µg/µL or 20 µg/µL protein solution can be tested to check capture response level.
Prepare other solutions with 1X running buffer.
Place the samples according to the series in autosampler.
Go to series in this method and start the capture step.
After the completion of this step, evaluate the capture protein response from the Measurements section in the project tree.
| A | B | C | D | E | |
| Activation | NiCl2 | 500 uM | 420 | 10 | |
| Wash (2x times) | Running buffer | 60 | 10 | ||
| Activation | EDC/NHS | 1:1 mix from 20C aliquots | 420 | 10 | |
| Capture | His tagged protein | [Y] | [Z] | 10 | |
| Passivation | Ethanolamine | 1 M | 420 | 10 | |
| Wash | Running buffer | 60 | 10 | ||
| Conditioning | EDTA | 350 mM | 420 | 10 |
A:Step, B:Solution, C:concentration, D:injection Duration, E:Flow rate (uL/min)
Once the evaluation and calculation are done, perform the immobilization assay for His tagged protein.
Perform Immobilization assay within the Measure tab.
First select the type of immobilization as Capture and amine coupling of His-tagged ligands to the Ni-NTA surface.
Select the Flow path as FC1 and FC2 (can be added FC3 and FC4 for different immobilization levels) for activation step.
Select FC2 for the Ni-NTA capture flow path and enter the details for protein concentration and injection duration(s)
FC1 is used as reference “blank” channel. It should not capture any protein.
Select FC1 and FC2 paths for Passivation step (FC3 and FC4 if they are included) .
Click Create Series.
Go to Autosampler section in the method. Check all the solution and their locations in the specified vial or plate.
Go to Cycles tab in the method and check activation (NiCl2 and EDC/NHS) and passivation, conditioning injections for only 1 and 2 otherwise all FCs are not included in the wizard. If they are included automatically, select only FC1 and FC2.
Place the samples according to the series in autosampler.
| 1 | 2 | 3 | 4 | 5 | |
| A | running buffer | ||||
| B | Protein solution | ||||
| C | EDC/NHS | NiCl2 | |||
| D | Ethanolamine | ||||
| E | EDTA | ||||
| F |
| 6 | 7 | 8 | |
| A | |||
| B | |||
| C | |||
| D | |||
| E | |||
| F |
Go to series in this method and start the immobilization.
Check the sensogram in real time through Measurement tab in the Project Tree.
Immobilization sensogram with His capture +amine coupling strategy
Creoptix relies on refractive index changes. Even the running buffer is used to dilute and prepare the solution in the immobilization step, there are still some variations the immobilization step. The final response levels might indicate near zero and/or below zero response as seen in the previous image (FC3:Green line). In this case, take protein capture level as a response.
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.
Acknowledgements