Biomolecule interactions including protein-protein and protein-DNA interactions are essential in biochemistry research. In this study we use biolayer interferometry, or BLI, an efficient technique for analyzing these interactions. We focus on replication protein A and its binding affinity for a single strand DNA to showcase the principles and capabilities of BLI.
Biolayer interferometry is an easy and cost-effective method to study protein kinetics. It allows quick real-time monitoring of protein interactions and offers a straightforward workflow with less complexity than other techniques. In our lab, we study the impact of post-translational modifications on the structure and function of the proteins that are essential for maintaining genome stability.
These modifications can initiate cascading effects across replication and repair pathways. By studying protein interactions, we aim to uncover the underlying mechanisms that safeguard genome integrity. To begin, prepare 12.5 nanomolar of three prime bio single-stranded poly dT 32 bait substrate and four concentrations of RPA using BLI buffer.
Using a pipette, add 250 microliters of BLI buffer in two 0.5 milliliter microfuge tubes and label them as AND. In a separate 96-well plate, pipette 200 microliters of BLI buffer in one well. Position the tray of biosensors on the 96-well plate such that one biosensor dips in the BLI buffer.
On the software, click on the hydrate option and set the timer to 10 minutes. Adjust the run settings for each step, baseline, association, and dissociation. Hit run on the software and follow the instructions in the prompt message.
Place two bay in the tube holder and attach the hydrated biosensor to the BLI system. Slide the tube holder under the biosensor at position A and monitor the baseline for the biosensor. After completing the initial baseline, open the lid and add four microliters of BLI buffer to the drop holder.
Slide the drop holder to the right under the same biosensor at position B.Once the lid is closed, monitor the BLI curve on the software. Then open the lid and replace tube A with tube D.Slide the new tube under the biosensor at position A before closing the lid and analyze the dissociation step in the interface. Place tube A into the tube holder.
Attach the hydrated biosensor to the BLI system and slide the tube holder under the biosensor as shown earlier. Then click run on the software interface for baseline setup. After adding four microliters of 12.5 nanomolar single-stranded DNA bait to the drop holder and sliding it under the biosensor for the association step, monitor the BLI curve on the software.
Then open the lid to replace tube A with D.Slide it under the biosensor and proceed with the dissociation step. Once the dissociation step is completed, open the lid and detach the biosensor. Then place the biosensor in the tray containing BLI buffer.
Using a pipette, add 250 microliters of BLI buffer to 10 0.5 milliliter black microfuge tubes. Label the tubes as a A1 to A5 and D1 to D5.Fill in the experimental details on the software interface and press the calculation button to process the information. In a separate 96-well plate pipette 200 microliters of stripping buffer into the well corresponding to the position of the hydrated biosensor.
After placing tube A1 in the tube holder, attaching the bait coated biosensor to the BLI system and sliding the tube under the biosensor, press run on the software. Once the initial baseline step is complete, open the lid and add four microliters of BLI buffer to the drop holder. Slide the drop holder under the biosensor and monitor the association step on the BLI interface.
Replace tube A1 with D1.Slide it under the biosensor and close the lid. Then monitor the dissociation step on the BLI interface. Once the dissociation step is complete, open the lid, remove the biosensor and place it back in its tray containing BLI buffer.
Then attach the bait-coated biosensor and place tube A2 in the tube holder. Slide the tube under the biosensor and click run on the software. After the baseline step, add four microliters of five nanomolar RPA onto the drop holder.
Slide the drop holder under the biosensor and close the lid. Monitor the association curve on the software interface. Remove the biosensor and dip it into the stripping buffer for 30 seconds.
Then dip the biosensor into the BLI buffer for three minutes. In the table labeled Run List, check the box reference for the analyte concentration of zero nanomolar to serve as a reference curve, check the box, analyze, for all concentrations of the analyte. Select both start of association and start of dissociation for step correction, choose global fitting for the assay.
Next, click analyze to process the data. To export the fitted data, right click on the graph generated from the analyze data. Select export data, then choose text or data, click on file and then browse to save the data in the DAT format.
Open the software and select advanced kinetics from the left panel. After placing a 96-well plate in the biosensor tray and adding 200 microliters of BLI buffer to five wells, insert the biosensor tray into the plate to hydrate the sensors. While the biosensors are hydrating, pipette 250 microliters of BLI buffer into 10 0.5 milliliter black centrifuge tubes.
Fill in the experimental details on the software interface and press the calculation button to process the information. After sliding the tube holder with tube A1 under the biosensor, click run to obtain the initial baseline. Add four microliters of the BLI buffer to the drop holder.
Slide the drop holder under the biosensor, close the lid and monitor the curve for loading in the software. The spectral shift during RPA binding using basic kinetics demonstrated a dose-dependent increase in binding strength with saturation achieved at 40 nanomolar. The spectral shift during RPA binding using advanced kinetics similarly showed dose-dependent binding, but with individual biosensors used per concentration for improved accuracy.
