Frequently Asked Questions
General
What is BLI?
Bio-Layer Interferometry (BLI) is a label-free, biosensor technology that enables the real-time measurement of molecular interactions. The Octet instrument shines white light down the biosensor and collects the light reflected back. Learn more about BLI.
What kinds of biosensors are available?
ForteBio produces a range of proprietary biosensors for different biological applications. Find out what biosensors are currently being offered.
Can the Octet System read crude samples?
BLI readings using the Octet System are based on the proteins bound at the tip of the biosensor and are independent of the sample medium for most biological applications. Quantitation and kinetic measurements can be made in serum-containing media (up to 10%) DMSO-containing buffers, periplasmic fractions, uncleared cell culture supernatants, and uncleared bacterial lysates.
What sample concentration is required?
The sample concentration range required to make accurate measurements depends on the specifics of the interaction and the data required.
- A standard, automated human IgG quantitation measurement can be made using a one-minute read time and with <10% CV for samples between 1 µg/mL and 100 µg/mL.
- A typical kinetic interaction can be measured over the range of 1 mM to 5 pM.
How much sample is required?
A sample volume of 200 µL is required in the microplate well to make accurate measurements. Since the sample is analyzed non-destructively, it can be fully recovered from the microplate well.
What happens if sample volume is less than 200 µL?
Sample volumes less than 200 µL are not recommended because they may cause measurement artifacts due to internal reflections during the orbital agitation of the microplate well.
What kind of throughput can be achieved using the Octet System?
Because of the flexibility of the ForteBio Octet System, throughput depends on the nature of the particular analysis being performed.
- For an automated Human IgG quantitation assay, the Octet System uses a standard assay time of 60 seconds for up to eight samples in parallel. A full plate of 96 samples can be assayed and the data fully analyzed in less than 20 minutes.
- For automated off-rate screening using a five-minute dissociation window, a 96-well microplate can be analyzed in one hour.
- For full kinetic analysis, eight samples can be processed in parallel with up to three-hour total assay times.
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Quantitation
How can nonspecific binding be prevented?
Nonspecific binding is minimized by a protein layer coating on the active surface of ForteBio biosensors. This protein layer enables reproducible attachment of the capture molecules and allows binding proteins to maintain their native binding properties by preventing direct interaction with the sensor surface. Learn more about the BLI technology at the core of ForteBio biosensors.
Kinetics
How many binding sites are on the biosensor?
ForteBio biosensors are coated with a protein layer to enable reproducible attachment of the capture molecules and minimize nonspecific binding. The protein conformation provides approximately 10e9 capture sites per sensor.
How can diffusion effects be avoided?
To overcome the effects of diffusion on kinetic measurements, the sample plate is subject to orbital motion relative to the biosensor. Experiments can be performed with static samples (for loading steps), or with motion ranging from 100 to 1,500 rpm.
Why does the y-axis display thickness?
The real-time kinetic display of Octet raw data represents changes in the binding to the surface of the biosensor. Binding to the biosensor surface results in an average optical thickness change expressed in nm.
How quickly can off-rates and on-rates be measured?
Rapid kinetic measurements on the Octet System are limited by the data-sampling rate. In practice, accurate measurements for protein kinetics can be made for rates which proceed to completion in as little as 20 seconds.
What is the slowest measurable off-rate?
Off-rate measurements in the Octet can be made over an extended time period. The primary limitation is sample evaporation from the microplate wells. For a typical experiment at 30°C using 200 µL well volume, the dissociation can be monitored for about three hours.
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