Biolayer Interferometry Technology to Detect Interactions between Ligand and Analyte

Protocolo

1. Biosensor hydration and assay set-up

1. Turn the BLItz machine on.
2. Ensure that the machine is connected to the computer through a USB data output port at the back of the machine.
3. On the computer, open the associated software (e.g., BLItz Pro), and click on Advanced Kinetics on the left-hand side of the screen.
4. On the software, type out all appropriate information about the experiment (including the Experiment Name, Description, Sample ID, and Protein Concentration) under each respective heading.
5. Click on Biosensor Type and choose Ni-NTA from the drop-down menu.
   1. Under the Run Settings heading, verify that the Shaker is set to Enable.
   2. Under the Step Type List heading, verify that there are 5 items listed: Initial Baseline, Loading, Baseline, Association, and Dissociation.
      NOTE: The duration of each step can be changed from default as needed. For optimal results, use a minimum of 30 s for Initial Baseline and Baseline; and 120 s for Association and Dissociation. The duration of the Loading step (ranging from 120 to 240 s) will depend upon the concentration of the ligand and affinity of the His-epitope tag on the ligand to the Ni-NTA-biosensor.
6. Remove the hydrated Ni-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.
   NOTE: Do not let the biosensor dry out during the experiment.
7. Place a 0.5 mL black microcentrifuge tube into the tube holder of the machine and pipette 400µL of the BLI buffer into it.
8. Verify that the slider of the machine is positioned such that the tube holder is situated in front of the black arrow on the machine.
9. Close the cover of the machine such that the biosensor tip becomes submerged in the buffer in the microcentrifuge tube.
10. Click Next on the software to begin recording the Initial Baseline.

2. Loading of ligand onto biosensor

1. After the Initial Baseline step has finished recording, open the cover of the machine.
2. Move the slider to the right such that the drop holder (instead of the tube holder) is situated in front of the black arrow.
3. Pipette 4 µL of a dialyzed His-tagged ligand onto the drop holder and close the cover of the machine.
   NOTE: The optimal concentration of the ligand to be used may vary for each protein. A concentration between 1.0 to 2.0 mg/mL is usually adequate to saturate the NTA at the tip of the biosensor in 240 s.
4. On the software, click Next to begin Loading.

3. Washing away additional ligand

1. After the Loading step has finished recording, open the cover of the machine.
2. Move the slider to the left such that the tube holder is once again situated in front of the black arrow.
3. 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.
4. Click Next once again on the software to begin recording the Baseline.

4. Association of analyte to ligand

1. After the Baseline step has finished recording, open the cover of the machine.
2. Remove the drop holder and clean it by pipetting out any protein and rinsing it with double-deionized water (ddH₂O) for a total of 5 times.
   1. Use a tissue wipe to clean the surface of the drop holder after the wash.
3. Replace the drop holder back onto the machine.
4. Move the slider on the machine to the right such that the drop holder is once again situated in front of the black arrow.
5. Pipette 4 µL of a dialyzed analyte onto the drop holder and close the cover of the machine.
6. On the software, click Next to begin Association.

5. Dissociation of analyte from ligand

1. After the Association step has finished recording, open the cover of the machine.
2. Move the slider on the machine to the right such that the tube holder is once again situated in front of the black arrow.
3. On the software, click Next to begin Dissociation.
4. After the Dissociation step has finished recording, open the cover of the machine.
5. Remove the drop holder and tube holder.
6. Rinse both with ddH₂O thoroughly to wash away any protein.
7. Remove the biosensor and discard it safely.

Materiais

Name	Company	Catalog Number	Comments
BLItz machine	ForteBio	45-5000	Device
Dip and Read Ni-NTA biosensor tray	ForteBio	18-5101	Ready-to-use Ni-NTA biosensors for poly-His-tagged Proteins
Drop holder	ForteBio	45-5004	Device
PCR tubes (0.2 mL)	Thomas Scientific	CLS6571	Plastic ware
Microcentrifuge tubes (black)	Thermo Fisher Scientific	03-391-166	Plastic ware
Kimwipes	Thermo Fisher Scientific	06-666A	Plastic ware
DTT	Thermo Fisher Scientific	R0861	Chemical
EDTA	MilliporeSigma	E6758	Chemical
MgCl2	MilliporeSigma	M8266	Chemical
NaCl	MilliporeSigma	S9888	Chemical
Tris-HCl	GoldBio	T095100	Buffer