Protein-protein interaction in transcription has been traditionally studied using Por dialysis. However, Por dialysis are purely quantitative. We use biolayer interferometry, or BLI, to overcome this problem.
Compared to Por dan, BLI detects real time association and dissociation between bonding partners. It also generates quantitative kinetic parameters, which are indicative of interaction mechanisms. BLI technology may sound intimidating, but it's not difficult to learn To use this technology, one has to have the access to a BLI instrument and the associated software.
This video will enable you to easily adapt to the BLI technology. Approximately 10 minutes prior to the start of an assay, pipette 200 microliters of the BLI buffer into a PCR tube. Remove a nickel NTA biosensor from the original packaging by holding the wide portion of the biosensor using a gloved hand.
Place the biosensor over the PCR tube such that only the glass tip of the biosensor is submerged in the BLI buffer. Keep the biosensor tip submerged for at least 10 minutes to ensure full hydration. Turn on the Blitz machine.
Ensure that the machine is connected to the computer through a USB data output port at the back of the machine. On the computer, open the associated software and click on Advanced Kinetics on the left hand side of the screen. In the software, type out all appropriate information about the experiment under each respective heading.
Click on Biosensor Type and choose Nickel NTA from the dropdown menu. The duration of each step can be changed from default as needed. For optimal results use a minimum of 30 seconds for initial baseline and baseline and 120 seconds for association and dissociation.
Remove the hydrated nickel NTA biosensor from the PCR tube and affix it to the biosensor mount on the machine by sliding the wide portion of the biosensor onto the mount. Place a 0.5 milliliter black microcentrifuge tube into the tube holder of the machine and pipette 400 microliters of BLI buffer into it. Close the cover of the machine such that the biosensor tip becomes submerged in the buffer in the microcentrifuge tube.
Click Next on the software to begin recording the initial baseline. After the initial baseline step has finished recording, open the cover of the machine. Move the slider to the right, such that the drop holder is situated in front of the black arrow.
Pipette four microliters of a dialyzed His-tagged ligand onto the drop holder and close the cover of the machine, which will automatically begin recording the loading step. After the loading step has finished recording, open the cover of the machine. Move the slider to the left, such that the tube holder is once again situated in front of the black arrow.
Close the lid of the machine and ensure that the biosensor tip is submerged into the BLI buffer of the tube in the tube holder. The machine and software will automatically begin recording the baseline step. After the baseline step has finished recording, open the cover of the machine.
Remove the drop holder and clean it by pipetting out any protein and rinsing it with double deionized water for a total of five times. Use a tissue wipe to clean the surface of the drop holder after the last wash. Replace the drop holder back onto the machine.
Move the slider on the machine to the right, such that the drop holder is once again situated in front of the black arrow. Pipette four microliters of a dialyzed analyte onto the drop holder and close the cover of the machine, which will automatically begin recording the association step. After the association step has finished recording, open the cover of the machine.
Move the slider on the machine to the right, such that the tube holder is once again situated in front of the black arrow and close the cover of the machine, which will automatically begin recording the dissociation step. After the dissociation step has finished recording, open the cover of the machine and remove the drop holder and tube holder. Thoroughly rinse both with double deionized water to wash away any protein.
Remove the biosensor and discard it safely. Repeat these steps for the same ligand analyte pair using different analyte concentrations. Once all runs have finished, save the data on the software by clicking File and Save Experiment As on the left side of the screen.
Under the Run Data heading, select Step Correction and Fitting one to one and click Analyze to generate kinetic data. To extract the quantitative data into a worksheet and generate graphs, click on Export to CSV and save the recorded data as a CSV file. Open the CSV file using a spreadsheet software.
Full length GrgA is made of 288 amino acids. As shown here, a 28 amino acid middle region binds Sigma 28 directly. Here the middle region tagged with an end terminal His-tag was used as the ligand, which was first immobilized to the tip of a nickel NTA biosensor.
Recordings of experiments with three different analyte concentrations each starting 30 seconds prior to ligand binding and ending two minutes after the beginning of the last wash are shown. Enhanced visualization of ligand analyte association and dissociation is shown following removal of values in the first two stages and resetting of the baseline. After washing unbound and terminal His-tagged GrgA 138 to 165 off the biosensor, real time association with the analyte was recorded following the addition of Sigma 28.
Finally, the real time dissociation was recorded following the wash. Before BLI assays, glycerol is removed from ligands and analytes. We recommend that BLI be performed soon after dialysis as discussed in the text.
After characterizing protein-protein interactions in transcription with BLI, one can investigate how the interaction affects transcription initiation, elongation, and or termination.
