Background on Molecular Interactions Using BLI
Some questions about BioLayer Interferometry and its application are answered below, and many more on the fortéBio web site, www.fortebio.com. To see some literature on the fortéBIO website, you need to register as a user with fortéBIO.
Amount of material bound on a probe
Sample volume in a well of a 96 well plate
Association rates that can be measured
Dissociation rates that can be measured
Dissociation constant range
Molecular weight range for analytes
Protein quantitation range for hIgG
ka 101 to 107 M-1 s-1
kd 10-6 to 10-1 s-1
10-3 to 10-11M
150 Da to 200 nm diamter
0.05 to 100 µg/mL (lower limits for some assays)
fortéBIO explained the technology and applications of their instruments in a Power Point presentation via Webex; click this link to open it.
What is BioLayer Interferometry?
What are the different methods for immobilizing a protein on a probe?
Note that probes have a limited life, six months is the length usually mentioned. For work not involving antibodies, the streptavidin is most popular, and Super Streptavidin is recommended for small molecules. Probes come in trays of 96 probes.
Can probes be reused?
Yes, but for some situations, it may not be worth the trouble fo finding regeneration conditions, and verifying that reusing the probe does not alter results.
Situations where fortebio suggest reuse are with Super Streptavin probes for small molecules where often there is complete dissociation within several minutes, and when performing some protein quantitation assays.
The ability to read 8 probes at one time can speed screening of regeneration conditions.
Which plates can be used for samples?
Only 96 well, black, flat bottom, polypropylene, 200 µL well, Greiner Cat. No. 655209
Is non-specific binding a potential problem?
Is mass transport a problem?
Yes. Controlled by limiting the amount of protein coupled to a probe and using a high shaking rate when dipping probes into the analyte solution.
How much analyte is consumed in a measurement?
A streptavidin sensor has about 109 capture sites, others may have fewer.