Biolayer Interferometry – FAQs
What is BLI
BLI or “biolayer interferometry” is a label-free biosensor technology that allows for real-time measurements of molecular interactions by detecting changes in interference patterns of white light reflected from the surface of fiber-optic sensor tips.
What parameters of a molecular interaction can be determined with BLI?
BLI directly enables the real-time determination of association rate (kon) and dissociation rate (koff) of a molecular interaction. From these rates, the overall affinity of the interaction (KD) can be calculated. The KD value can also directly be determined from the concentration-dependent formation of molecular complexes.
What is the slowest dissociation rate that can be measured?
The primary limitation for the measurement of very low dissociation rates is sample evaporation from the microplate wells.
What is the fastest association rate that can be measured?
The primary limitation for the measurement of very high association rates is the data-sampling rate. In practice, association phases can be measured that reach their plateau in as little as 20 seconds.
Which types of sensor tips are available?
Around 20 different sensor types are available, including sensors coated with streptavidin for capturing biotinylated ligands, specifically coated sensors for capturing all kinds of antibody molecules and fragments, sensors with amine-reactive coating, affinity sensors against His- or GST-tags, as well as sensors coated with protein A, protein G, or protein L. A full list of available sensor types is available from PALL FortéBio.
How many binding sites are on the surface of a sensor tip?
PALL FortéBio sensor tips feature a base protein coating in order to assist in reproducible ligand capturing and to minimize non-specific binding. The sensor tips approximately provide 109 capture sites.
How much sample is required for Octet assays?
With the Octet® K2 system and standard 96-well plates, a minimum sample volume of 180 µL is required in order to assure sufficient orbital flow around the tip. Importantly, the sample can be fully recovered from the plate after the measurement, because the sample is analyzed non-destructively. Sample volumes below 180 µL are not recommended, as they may lead to internal reflections in the 96-well plate.
What concentration should samples have?
The specific sample concentration required for precise measurements depends on sample type, interaction affinity, or the buffer or liquid used. For example, human IgG antibodies can be analyzed in concentrations between 50 ng/mL and 2000 µg/mL.
Is it possible to analyze complex and crude samples?
For most biological applications, BLI measurements are independent of the buffer composition or the composition of the sample medium. For example, small-molecules can easily be analyzed in buffers with high DMSO-concentrations. Moreover, complex biological liquids like serum, cell fractions and cell lysates, as well as bacterial lysates or cell culture supernatants can be analyzed.
Is non-specific binding a problem?
In general, the sensor tips from PALL FortéBio cotain a base protein layer on their surface that minimized most non-specific binding. This layer also enhances ligand capture by preventing them from directly contacting the sensor surface. If further reduction of potential non-specific binding is required, this can usually be achieved with blocking steps with BSA for example.
Why does the vertical axis of BLI plots display a thickness in nm?
BLI measures the changes in interference patterns of white light at the surface of a fiber-optic sensor tip. These changes in turn are a function of changes in the thickness (in nm) of the molecular layer on top of the sensor tip surface.