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. BLI detects changes in the interference pattern of white light reflected back from the surface of disposable fiber optic biosensors. Learn more about BLI.

What kinds of biosensors are available?

Pall ForteBio offers a range of biosensor chemistries for different biological applications. Find out what biosensors are currently being offered.

Can the Octet System read crude samples?

BLI measurements using Octet systems 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, 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 sample type, the specific interaction, as well as on the instrument type (Octet or BLItz system) used.

For example: A standard human IgG quantitation measurement can be made using Protein A biosensors in a 2-minute incubation for samples between 50 ng/mL and 2000 µg/mL on the Octet RED96 and Octet RED384 systems. For best results, we recommend operating within the linear dynamic range, which is molecule-specific and need to be defined for your assay.

How much volume of sample is required for octet assays?

A minimum sample volume of 180 µL is required in a standard 96-well microplate to make accurate measurements. In a standard 384-well microplate, the sample volume required is 120 µL, and in a tilted-bottom 384TW microplate, the minimum required volume is 40 µL. Since the sample is analyzed non-destructively, it can be fully recovered from the microplate well. For details on recommended microplates for Octet instruments, please refer to What are the recommended microplates for Octet instruments?

What happens if sample volume is less than 180 µl in a 96-well microplate on the Octet systems?

Sample volumes less than 180 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 Pall ForteBio Octet systems, throughput depends on the nature of the particular analysis being performed and the Octet instrument model that is used.

For a high-throughput, automated human IgG quantitation assay on the Octet HTX system, a full plate of 96 samples can be assayed and the data fully analyzed in 2 minutes. For automated off-rate screening, a 96-well microplate can be analyzed in a few minutes rather than hours. Please see the datasheets for our other Octet systems for throughput specifications:

Quantitation

How can nonspecific binding be prevented?

All ForteBio biosensors with the exception of AR and AR2G contain a protein layer on the surface of ForteBio biosensors that minimizes most non-specific binding events. 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. Further reduction in non-specific or undesirable binding events can potentially be achieved by performing an blocking step using an agent such as BSA.

Kinetics

How many binding sites are on the biosensor?

Pall ForteBio biosensors are coated with a protein layer to enable reproducible attachment of the capture molecules and minimize non-specific binding. The protein conformation provides approximately 109 capture sites per biosensor.

How can diffusion effects be avoided on the Octet systems?

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 or BLItz raw data represents changes in the binding to the surface of the biosensor. The Y-axis measures the wavelength change in BLI interference peaks (in nm), which is a function of changes to the average optical thickness.

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.