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.

Disclosures

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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Cite This Article
Graphene Biosensor-Based Field-Effect Biosensing to Detect Protein-Protein Interactions. J. Vis. Exp. (Pending Publication), e21392, doi: (2023).

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