Graphene Biosensor-Based Field-Effect Biosensing to Detect Protein-Protein Interactions
Published: May 31, 2023
Abstract
Source: Qvit, N., et al. Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology. J. Vis. Exp. (2022).
In this video, we describe the field-effect biosensing (FEB) technology to detect and quantify protein-protein interactions. The target proteins immobilized on the graphene biosensor interact with analyte proteins in solution, resulting in electrical current alterations in the FEB system. This change in the electrical response indicates the binding affinity between the proteins.
Protocol
NOTE: The recombinant proteins used in this study, Hsp90, and Cdc37, were commercially obtained (see Table of Materials). 1. Chip Activation NOTE: All materials to be used in the experiment are listed in the Table of Materials. Filter all prepared solutions through a sterile 0.2 μm filter. Prepare 1-Ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC) solution by adding 2 mg of EDC t…
Representative Results
Figure 1: Protocol summary. (A) Summary of the steps for the chip activation process. (B) Graphical representation of the five repeating steps in the protocol.
Declarações
The authors have nothing to disclose.
Materials
| Automated analysis software | Agile plus software, Cardea (Nanomed) | NA CAS number: NA |
Referred to in the text as the automated analysis software supplied with the instrument. Generates automated analysis. |
| COOH-BPU (Biosensing Processing Unit) | Agile plus software, Cardea (Nanomed) | NA CAS number: NA |
Biosensor chip |
| Data review software | Datalign 1.0, Cardea (Nanomed) | NA CAS number: NA |
Referred to as the supplied data review software in the text. Supplied with the instrument and allows to review and export the information data points. |
| Dialysis bag | CelluSep, Membrane filtration products | T2-10-15 CAS number: NA |
T2 tubings (6,000-8,000 MWCO), (10 mm fw, 6.4mm Ø, 0.32ml/cm, 15m) |
| EDC (1-Ethyl-3-(3-dimethylamino propyl) carbodiimide) | Cardea (Nanomed) | EDC160322-02 CAS number: 25952-53-8 |
White powder |
| MES (2-(N-morpholino) ethane sulfonic acid) buffer | Merck | M3671-50G CAS number: 4432-31-9 |
White powder |
| NHS (N-Hydroxysulfosuccinimide) chips | Cardea (Nanomed) | NA CAS number: NA |
Graphene-based chip |
| PBS (Phosphate-buffered saline) X 10 | Bio-Lab | 001623237500 CAS number: 7758-11-4 |
Liquid transparent solution |
| Pipette | Thermo Scientific | 11855231 CAS number: NA |
Finnpipette F3 5-50 µL, yellow |
| Quench 1 (3.9 mM amino-PEG5- alcohol in 1 X PBS) | Cardea (Nanomed) | 0105-001-002-001 CAS number: NA |
Liquid, transparent solution |
| Quench 2 (1 M ethanolamine (pH=8.5)) | Cardea (Nanomed) | 0105-001-003-001 CAS number: NA |
Liquid, transparent solution |
| Recombinant protein Cdc37 | Abcam | ab256157 CAS number: NA |
|
| Recombinant protein Hsp90 beta | Abcam | ab80033 CAS number: NA |
|
| Spreadsheet | Excel, Microsoft office | NA CAS number: NA |
|
| Statistical software | GraphPad, Prism | NA CAS number: NA |
Referred to as the other statistical software. Sigma plot, phyton or other statistical programes may also be used |
| Sulfo-NHS | Cardea (Nanomed) | NHS160321-07 CAS number: 106627-54-7 |
White powder |
| Tips | Alex red | LC 1093-800-000 CAS number: NA |
Tip 1-200 µl, in bulk, 1,000 pcs |
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Citar este artigo
Graphene Biosensor-Based Field-Effect Biosensing to Detect Protein-Protein Interactions. J. Vis. Exp. (Pending Publication), e21392, doi: (2023).