Bio-Layer Interferometry SOP
To download a PDF of this SOP, click here.
- Get the sensor tips you’ll need pre-soaking with a black plate inside the extra tip tray. Let them soak for at least 10 minutes in 200 ul of buffer. Put the whole tray (minus the lid) into the Octet on the left platform and close the door.
- Fill the wells you’ll be using in a second black plate. (Draw a plate map first if that will help.)
- Put that plate into the Octet on the right platform, being sure it’s seated inside the white clips. Close the Octet door and pull down the In Use sign.
- Turn on the computer.
- In the ForteBio folder on the desktop, make a data folder for yourself.
- Open the Data Acquisition software and let the instrument initialize completely before opening the door. (Check instrument status in the small window on the lower left.)
- Choose whether you’re doing Quantitation or Kinetics. You can choose an existing method from this first window.
- On the Plate Definition tab, R click a column number to define what those wells are. (buffer, sample, etc.) Enter the concentration of your samples as a molar concentration only. The software needs this info to be able to calculate a KD.
- You can label a Reference well here by L clicking on it individually then R clicking to define it as a reference. (Or you can wait until you’re analyzing data to define references.) Ideally, you’ll have a Buffer Only reference and an Analyte Only reference. This will be necessary for the software to calculate a good R2.
- On the Assay Definition tab, highlight a column then click the Add button to choose a new type of step. Build your assay step by step, a typical sequence being: baseline, loading, baseline, association, dissociation. To move the assay steps to the lower chart, double click on the arrow next to each step in the upper chart. Be sure sample number matches column number in the assay chart.
- You can change the length of time for each step but leave the rpm set at 1,000 for kinetics work. If you define the type of tip on this tab, it populates the next one. You can set a Threshold for the Loading step only. This will determine how much material you load onto the tip. A 1-2 nm shift is good to start with.
- On the Sensor Assignment tab, highlight the well at the top of the column then R click to choose the sensor type. Be sure that the column with your sensors has purple squares in it. Uncheck “Replace sensors in tray after use” to have them ejected into the waste tray at the end of the experiment.
- Review Experiment lets you walk through the sequence of events, using the arrow buttons. Check that the correct columns are chosen for each step and that the time interval is correct.
- On the Run Experiment tab, set the data path to your folder with the browse icon. (three small dots) Everyone has a personal folder in the desktop ForteBio folder. Then name your experiment in the second line. Uncheck Delayed Expt. Start. (usually) Then click the green GO button.
- During the experiment, you can alter the length of a step with the Go to Next Step and Extend Current Step buttons if it looks like things are taking too long or will be over too soon. Once the experiment is complete, the word DONE will appear in its title bar. Close the Data Acquisition software and go to the Data Analysis software.
Note: This protocol is a practical hands-on guide to using the instrument.
Reading this is not a substitute for reading the manufacturer's manual,
something we strongly recommend that you do.