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.
