Biolayer Interferometry Technology to Detect Interactions between Ligand and Analyte
Transcription
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 "Ni-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 microlitres 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. Pipette 4 microlitres 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 4 microlitres 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.
