This research was supported by a grant from the Binational Science Foundation (BSF) to S.K.S. and N.Q.

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 &#956;m filter.
<ol>
	<li>Prepare 1-Ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC) solution by adding 2 mg of EDC to 2.5 mL of 1 M 2-...

The authors declare no conflicts of interest, financial or otherwise.

In this study, the feasibility of using the FEB technology (a real-time kinetic characterization approach) was evaluated to determine the biomolecular interaction between Hsp90 and Cdc37. The initial exploratory experiment (first experiment) suggested that choosing the proper analyte concentrations is a critical part of the experiment and that the experiment should be designed by including concentration points above and below the KD value, which were predicted based on the data available in the literature....

Biomolecular interactions: 
Proteins are essential parts of organisms and participate in numerous molecular pathways such as cell metabolism, cell structure, cell signaling, immune responses, cell adhesion, and more. While some proteins perform their function(s) independently, most proteins interact with other proteins using a binding interface to coordinate proper biological activity1.
Biomolecular interactions can mainly be classified based on the distinct structural and functional characteristics of proteins involved2, for example, based on the protein surfaces, the complex stability, or the persistence of interactions3. Identifying essential proteins and their roles in biomolecular interactions is vital for understanding biochemical mechanisms at the molecular level4. Currently, there are various approaches to detect these interactions5:&#160;in vitro6, in silico7, in live cells8,&#160;ex vivo9, and in vivo10&#160;with each having its own strengths and weaknesses.
The in vivo assays are performed using the whole animal as an experimental tool11, and the ex vivo assays are performed on tissue extracts or whole organs (e.g., heart, brain, liver) in a controlled external environment by providing minimal alterations in natural conditions.&#160;The most common application of in vivo and ex vivo studies is to evaluate the pharmacokinetics, pharmacodynamics, and toxicity effects of potential pharmacological agents before human trials by ensuring their overall safety and efficacy12.
Biomolecular interactions can also be detected within living cells. Imaging live cells allow us to observe dynamic interactions as they execute the reactions of a particular biochemical pathway13. Moreover, detection techniques, such as bioluminescence or fluorescence resonance energy transfer, can provide information about where and when these interactions occur within the cell14. Although detection in live cells offers crucial details, these detection methodologies rely on optics and labels, which may not reflect the native biology; they are also less controlled than in vitro methods and require specialized expertise to perform15.
The in silico computational methods are primarily used for large-scale screening of target molecules before the in vitro experiments. Computational prediction methods, computer-based databases, molecular docking, quantitative structure-activity relationships, and other molecular dynamics simulation approaches are among the well-established in silico tools16. Compared to laborious experimental techniques, the in silico tools can easily make predictions with high sensitivity, but with reduced accuracy in predictive performance17.
In vitro assays are performed with microorganisms or biological molecules outside of their standard biological context. Portraying biomolecular interactions through in vitro methods is critical to understanding protein functions and the biology behind the complex network of cell functioning. The preferred assay methodology is chosen according to the protein&#39;s intrinsic properties, kinetic values, and the mode and intensity of interactions18,19.
The Hsp90/Cdc37 interaction: 
The chaperone-kinase pathway, connecting Hsp90 and Cdc37, is a promising therapeutic target in tumor biology20. Hsp90 plays a central role in cell cycle control, protein assembly, cell survival, and signaling pathways. Proteins that rely on Hsp90 for their functions are delivered to Hsp90 for complexation through a co-chaperone, such as Cdc37. The Hsp90/Cdc37 complex controls the folding of most protein kinases and serves as a hub for a multitude of intracellular signaling networks21. It is a promising anti-tumor target due to its elevated expression in various malignancies, including acute myeloblastic leukemia, multiple myeloma, and hepatocellular carcinoma22,23.
Commonly used in vitro biomolecular interaction detection techniques 
Co-immunoprecipitation (co-IP) is a technique relying on antigen-antibody specificity to identify biologically relevant interactions24. The primary disadvantage of this method is its inability to detect low-affinity interactions and kinetic values24. Biophysical methods such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), biolayer interferometry (BLI), and FEB technology are preferred to determine the kinetic values.
ITC is a biophysical detection method based on the determination of binding energy along with a complete thermodynamics analysis to characterize biomolecular interactions25. The primary advantage of ITC is that it does not require any labeling or fixation of the target protein. The main difficulties encountered by ITC are the high concentration of target protein required for one experiment and the difficulty in analyzing non-covalent complexes due to small binding enthalpies26. Both SPR and BLI are label-free biophysical techniques that rely on the immobilization of the target molecule on the sensor surface, followed by subsequent injections of the analyte over the immobilized target27,28. In SPR, alterations in the refractive index during biomolecular interactions are measured27; in BLI, the interference in reflected light is recorded in real-time as a change in wavelength as a function of time28. Both SPR and BLI share common advantages of offering high specificity, sensitivity, and detection capabilities29. In both methods, the target protein is immobilized on biosensor surfaces, and hence, there may be some loss of the native conformation of the target, which makes it difficult to discriminate between specific vs. non-specific interactions30. BLI uses expensive disposable fiber-optic biosensors to immobilize the target, and is, therefore, a costly technique31. Compared to these well-established biomolecular detection tools, FEB technology offers a reliable and label-free platform by using low nanomolar concentrations for biomolecular detection in real-time with kinetic characterization. The FEB technology also overcomes the bubbling challenges faced in ITC and is more cost-effective compared to SPR or BLI.
The field-effect transistor (FET) based biosensors is an emerging field for detecting biomolecular interactions by offering varied biomedical applications. In the FET system, targets are immobilized to the biosensor chips and interactions are detected by changes in conductance32. The unique feature to be considered in the development of an efficient electronic biosensor is the physicochemical properties such as the semi-conductive nature and chemical stability of the coating material used to fabricate the sensor surface33. Conventional materials like silicon used for FET have limited the sensitivity of sensors because it requires oxide layers sandwiched between the transistor channel and a specific environment for proper functioning34. Moreover, silicon transistors are sensitive to high salt environments, thus making it hard to measure biological interactions in their natural environment. The graphene-based biosensor is presented as an alternative as it offers excellent chemical stability and electric field. Since graphene is a single atomic layer of carbon, it is both extremely sensitive as a semi-conductor and chemically compatible with biological solutions; both of these qualities are desirable to generate compatible electronic biosensors35. The remarkable ultrahigh loading potential of biomolecules offered by graphene-coated biosensors lead to the development of graphene-based biosensors FEB technology.
Principle of FEB technology: FEB is a label-free biomolecular detection technique that measures the electric current through the graphene biosensor to which the binding targets are immobilized. Interactions between the immobilized protein and the analyte result in alterations in current that are monitored in real-time, enabling accurate kinetic measurements36.
Instrumentation: The FEB system comprises a graphene field-effect transistor (gFET) sensor chip and an electronic reader that applies a constant voltage throughout the experiment (Figure 1). The analyte is applied in solution to the target protein immobilized on the biosensor surface. When an interaction occurs, an alteration in the current is measured and recorded in real-time. As the analyte concentration increases, the fraction of bound analyte will also increase, causing higher alternations in the current. Using the automated analysis software provided with the instrument (Table of Materials), I-Response is measured and recorded in terms of biosensing units (BU)37. I-Response is defined as the alteration in the current (I) through the biosensor chip measured in real-time upon the interaction of the immobilized target with the analyte. The FEB automated analysis software can analyze both the I-Response and C-Response to dynamic interaction events, where the C-Response records the alterations in the capacitance (C). The variations in both the I-Response and C-Response correspond directly to the fraction of bound analyte and can be further analyzed to generate KD values. The automated analysis software&#39;s default preference is I-Response.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63495/63495fig01.jpg" /> 
Figure 1: Overview of the experimental setup. (A) Graphene-based chip and an electronic reader. (B) An overview of the chip components. The chip is attached to two electrodes that supply current to the system. The surface of the chip is covered with graphene, which when activated can bind the target. <a href="https://www.jove.com/files/ftp_upload/63495/63495fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Methodology: 
Initially, the activated biosensor chip is inserted into the FEB device (Figure 1) followed by the execution of the steps outlined below: (1) Calibration: The experiment starts with system calibration using 1x phosphate-buffered saline (PBS; pH = 7.4) to create the baseline equilibration response. (2) Association: The analyte is introduced in the chip, and the I-Response is monitored until binding saturation is reached. (3) Dissociation: The analyte is dissociated using 1x PBS. (4) Regeneration: Remnants of the analyte are removed using 1x PBS. (5) Washing: A total of five washes are performed using 1x PBS for the thorough removal of the bound and unbound analytes from the chip.
Analysis: 
Data analysis is performed using the fully automated software provided with the instrument. The automated analysis software generates a Hill fit plot with a KD value. The Hill fit plot describes the association of an analyte to the target protein as a function of analyte concentrations. The concentration at which a half-maximal response is achieved is proportional to the KD value. A low KD value represents high binding affinity and vice versa.
To validate the data obtained from the FEB experiment, I-Responses are extracted from each readout point for each analyte concentration using the data review/export software and can be exported to other statistical analysis software (see Table of Materials) as explained below.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automated analysis software</td>
			<td>Agile plus software, Cardea (Nanomed)</td>
			<td>NA 
			CAS number: NA</td>
			<td>Referred to in the text as the automated analysis software supplied with the instrument. Generates automated analysis.</td>
		</tr>
		<tr>
			<td>COOH-BPU (Biosensing Processing Unit)</td>
			<td>Agile plus software, Cardea (Nanomed)</td>
			<td>NA 
			CAS number: NA</td>
			<td>biosensor chip</td>
		</tr>
		<tr>
			<td>Data review software</td>
			<td>Datalign 1.0, Cardea (Nanomed)</td>
			<td>NA 
			CAS number: NA</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Dialysis bag</td>
			<td>CelluSep,&#160; Membrane filtration products</td>
			<td>T2-10-15 
			CAS number: NA</td>
			<td>T2 tubings (6,000-8,000 MWCO), (10 mm fw, 6.4mm &#216;, 0.32ml/cm, 15m)</td>
		</tr>
		<tr>
			<td>EDC (1-Ethyl-3-(3-dimethylamino propyl) carbodiimide)</td>
			<td>Cardea (Nanomed)</td>
			<td>EDC160322-02 
			CAS number: 25952-53-8</td>
			<td>White powder</td>
		</tr>
		<tr>
			<td>ITC (Isothermal titration calorimetry) system</td>
			<td>Microcal-PEAQ-ITC (Malvern, United Kingdom)</td>
			<td>NA 
			CAS number: NA</td>
			<td></td>
		</tr>
		<tr>
			<td>MES (2-(N-morpholino) ethane sulfonic acid) buffer</td>
			<td>Merck</td>
			<td>M3671-50G 
			CAS number: 4432-31-9</td>
			<td>White powder</td>
		</tr>
		<tr>
			<td>NHS (N-Hydroxysulfosuccinimide) chips</td>
			<td>Cardea (Nanomed)</td>
			<td>NA 
			CAS number: NA</td>
			<td>Graphene-based chip</td>
		</tr>
		<tr>
			<td>PBS (Phosphate-buffered saline) X 10</td>
			<td>Bio-Lab</td>
			<td>001623237500&#160; 
			CAS number: 7758-11-4</td>
			<td>Liquid transparent solution</td>
		</tr>
		<tr>
			<td>Pipete</td>
			<td>Thermo Scientific</td>
			<td>11855231 
			CAS number: NA</td>
			<td>Finnpipette F3 5-50 &#181;L, yellow</td>
		</tr>
		<tr>
			<td>Quench 1 (3.9 mM amino-PEG5-alcohol in 1 X PBS)</td>
			<td>Cardea (Nanomed)</td>
			<td>0105-001-002-001 
			CAS number: NA</td>
			<td>Liquid, transparent solution</td>
		</tr>
		<tr>
			<td>Quench 2 (1 M ethanolamine (pH=8.5))</td>
			<td>Cardea (Nanomed)</td>
			<td>0105-001-003-001 
			CAS number: NA</td>
			<td>Liquid, transparent solution</td>
		</tr>
		<tr>
			<td>Recombinant protein Cdc37</td>
			<td>Abcam</td>
			<td>ab256157 
			CAS number: NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant protein Hsp90 beta</td>
			<td>Abcam</td>
			<td>ab80033 
			CAS number: NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet</td>
			<td>Excel, Microsoft office</td>
			<td>NA 
			CAS number: NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Statistical software</td>
			<td>GraphPad, Prism</td>
			<td>NA 
			CAS number: NA</td>
			<td>Referred to as the other statistical software. Sigma plot, phyton or other statistical programes may also be used</td>
		</tr>
		<tr>
			<td>Sulfo-NHS</td>
			<td>Cardea (Nanomed)</td>
			<td>NHS160321-07 
			CAS number: 106627-54-7</td>
			<td>White powder</td>
		</tr>
		<tr>
			<td>Tips</td>
			<td>Alex red</td>
			<td>LC 1093-800-000 
			CAS number: NA</td>
			<td>Tip 1-200 &#181;l, in bulk, 1,000 pcs</td>
		</tr>
	</tbody>
</table>

exploring biomolecular interaction between the molecular chaperone hsp90 and its client protein kinase cdc37 using field-effect biosensing technology

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 &#181;M, 0.053 &#181;M, and 0.072 &#181;M in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.

Results from experiment 1: 
The target protein Hsp90 (500 nM) was immobilized to the chip following the target immobilization protocol as described above. For the first experiment, 10 concentrations of the analyte protein, Cdc37, ranging from 25 nM to 5,000 nM, were prepared based on the data available in the literature (see Table 1).
The steps of the experiment can be monitored in real-time by following the alterations occurring in the I-Response (<stron...

Watch this Scientific Journal Video about Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology at JoVE.

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...

FEB is a label-free and cost-effective ______ _______ interaction detection platform which offers fast and accurate measurements. This method offers validation of known or unknown interactions and an easy way to test for potential inhibitors of the biomolecular interactions. The main advantage of FEB is its automated, user-friendly software that gates the user throughout the experiment with open, hand pipetting platform, enabling users to work with little or even no training.FEB technology also offers applications in diverse fields of pharmaceuticals, biomolecular analysis of small molecules, peptides and proteins, drug validation by translating in vitro biological activity readily into in vivo efficacy. Being a beginner, you may face challenges in choosing the right analyte concentrations to optimize the experiment to get a reliable KD value. We advise giving attention to the chip functionalization process and proper buffer exchange.Begin by mixing equal volumes of EDC and sulfo-NHS solution by pipetting up and down. Place the biosensor chip supplied by the company in a glass Petri dish with a fitted lid. All the functionalization steps involved in chip activation are suggested to be done within the Petri dish.Apply 50 microliters of one-molar MES buffer to the biosensor chip. Incubate for one minute at room temperature. Then, aspirate the buffer and apply 50 microliters of EDC sulfo-NHS solution immediately to the sensor chip.Cover the Petri dish and incubate for 15 minutes at room temperature. Aspirate the EDC sulfo-NHS solution from the chip and apply 50 microliters of one-molar MES buffer. Aspirate MES buffer and rinse the chip twice with 50 microliters of 1X PBS.Aspirate the PBS from the chip and add the target molecule Hsp90. Cover the glass Petri dish and incubate for 30 minutes at room temperature. Then, aspirate the solution containing the target molecule and rinse three times with 50 microliters of 1X PBS.Aspirate the 1X PBS solution from the chip and add 50 microliters of the quench one solution. Cover the glass Petri dish and incubate for 15 minutes at room temperature. Aspirate the quench one solution from the chip and add 50 microliters of the quench two solution.Cover the glass Petri dish and incubate for 15 minutes at room temperature. After that, aspirate the quench two solution from the chip and rinse the chip five times using 50 microliters of 1X PBS leaving the last PBS droplet on the sensor. Prepare the analyte dilution series for Cdc37 in the desired concentration range.Design the experiment to include at least eight different analyte concentrations to obtain a reliable dissociation constant, or KD value, and prepare these different dilutions in the same buffer used for calibration and target protein. Place the chip carefully to the instrument. Make sure the software is on and the LED light turns green.Next, press the Run Experiment module on the automated software and choose 10 points with regeneration or any other desired protocol. Fill in the details such as operator name, experiment name, date, regeneration buffer, immobilized target, and the analyte in solution. Press the Begin Experiment button displayed on the software and follow the instructions shown by the automated software.To perform the instrument calibration, aspirate the remaining PBS solution from the chip and apply 50 microliters of the calibration buffer. Press the Continue button and wait for five minutes until the calibration step is finished. The software displays the endpoint determined for the calibration step with a warning alarm to follow up.Next, perform an analyte association by aspirating the calibration buffer from the chip and applying 50 microliters of the lowest analyte concentration. Press the Continue button and wait for five minutes until the association step is finished. The software displays the end point for the association step with a warning alarm to proceed.To perform an analyte dissociation, aspirate the analyte solution from the chip and apply 50 microliters of the dissociation buffer. Press the continue button. After the dissociation step duration, the software displays the endpoint for the dissociation step with a warning alarm.Next, perform chip regeneration by aspirating the dissociation solution from the chip and applying 50 microliters of the regeneration buffer. Then, press the Continue button. The regeneration step typically takes approximately 30 seconds to finish.After that, the software displays the end point for the regeneration step with a warning alarm to follow up. Finally, to wash the chip, aspirate the regeneration solution from the chip and apply 50 microliters of the wash buffer. Aspirate the solution from the chip and repeat this five times.Leave the last drop of the wash buffer on the chip. Then, press the Continue button and wait for 30 seconds until the wash step duration is finished in the software display. Repeat the steps for each analyte concentration used.The five steps of the calibration, analyte association, dissociation, regeneration, and washing five times constitute one cycle. Press the Analysis button in the automated analysis software at the end of the experiment. A display window containing all the experimental points will appear.Ensure that the analyte concentrations used for the prescribed protocol are correct. Press the Run Analysis button to generate the KD value automatically. The software generates a hill fit plot by plotting the analyte concentrations against the corresponding I-responses from which the dissociation constant at equilibrium is calculated.The target protein Hsp90 was immobilized to the chip, and for the first experiment, 10 concentrations of the analyte protein Cdc37 ranging from 25 nanomolars to 5, 000 nanomolars were prepared. The data files generated from experiment one has been shown here. The experimental data monitored in real time is shown here.The Y-axis corresponds to the I-response in the biosensor units, and the X-axis corresponds to the different time points and concentrations of the analyte in the experiment. The graphical image shown here represents the hill fit plot generated from the automated analysis software. The Y-axis corresponds to the I-response in the biosensor units, and the X-axis corresponds to the different analyte concentrations in the experiment.The graphical image shown here represents the association plot generated using the statistical analysis software. The Y-axis corresponds to the I-response at the end of the association phase, and the X-axis corresponds to the different concentrations of the analyte Cdc37. In experiment two, a different set of concentrations ranging from 0.4 nanomolar to 200 nanomolar was used, and the generated data files are shown here.The graphical images represent the ITC thermogram of Hsp90 and Cdc37 interaction. Shown here are the corresponding heat-evolving curves obtained due to the consecutive injections of Cdc37 into Hsp90 at 298.15 Kelvin. The differential power used here is a function of time.The integrated data points in this plot designate the corresponding normalized heat versus the Mueller ratio of Hsp90 to Cdc37. The open pipetting approach used here requires that the user have a basic mastery of hand pipetting, making sure not to touch the biosensor with the tip. The reproducibility of the step timing depends entirely on the user.Once the researcher characterizes an interaction between two biomolecules, we can perform a screening experiment by designing potential inhibitors or modulators targeting the specific biomolecular interaction using the FEB system. This procedure can be used to identify, verify, and quantify known and novel interaction between proteins, as well as small molecules, including drugs and peptides.

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Research

JoVE Journal

Biochemistry

3.1K Görüntüleme.  Bar-Ilan University. FEB, hızlı ve doğru ölçümler sunan, etiketsiz ve uygun maliyetli bir __ ___ Bu yöntem, bilinen veya bilinmeyen etkileşimlerin doğrulanmasını ve biyomoleküler etkileşimlerin potansiyel inhibitörlerini test etmenin kolay bir yolunu sunar. FEB'in temel avantajı, açık, elle pipetleme platformu ile deney boyunca kullanıcıyı aradan geçiren ve kullanıcıların çok az veya hiç eğitim almadan çalışmasını sağlayan otomatik, kullanıcı dostu yazılımıdır.FEB tek...

Video: Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology