Biolayer Interferometry (BLI)

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BLI at the CMI

Bio-Layer Interferometry (BLI) is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip.  BLI experiments are used to determine the kinetics and affinity of molecular interactions. In a BLI experiment, one molecule is immobilized to a Dip and Read Biosensor and binding to a second molecule is measured. A change in the number of molecules bound to the end of the biosensor tip causes a shift in the interference pattern that is measured in real-time. 

BLI Samples         ForteBio Octet Red384ForteBio BLItz

The CMI has two ForteBio instruments for measuring BLI: the Octet RED384 and the BLItz.  The Octet RED384 is more sensitive and higher throughput and can be used for macromolecular and small molecule binding experiments.  The BLItz uses a very small sample volume, but is more time consuming and cannot measure molecules smaller than 10 KDa.

                 

Applications and Features

Applications

  • Kinetic binding: ka, kd
  • Equilibrium binding: KD
  • Macromolecular and small molecule binding (Octet only)

Octet RED384 Key Features

  • Simultaneous real-time binding on up to 16 biosensors
  • Disposable biosensors (sensor regeneration not required)
  • 96 or 384 well reagent plate format
  • Small molecule sensitivity

BLItz Key Features

  • Low sample volume: 4 μl sample
  • Disposable biosensors (sensor regeneration not required)

Theory and Design

 In a BLI experiment, one molecule is immobilized to a Dip and Read Biosensor and binding to a second molecule is measured. A change in the number of molecules bound to the end of the biosensor tip causes a shift in the interference pattern that is measured in real-time. Sensors are moved from one well of a microplate to another to change solution from samples (load sample and analyte sample) to buffer for baselines and dissocation.

BLI_Sensors

Figure adapted from ForteBio

BLI Experimental Phases

There are two major phases in a BLI experiment and each will need to be optimized for you experiment. Regeneration is an optional third phase that may be used when a single set of sensors is reused for multiple measurements.

Immobilization

  • the Load molecule is attached irreversibly or reversibly to the sensor chip surface

Interaction analysis

  • In the association phase, the Analyte is injected over the sensor surface
  • In the dissociation phase, the Analyte is washed off the surface

Regeneration

  • the surface is regenerated by removing remaining bound analyte or by removing ligand (which will also remove remaining analyte)

BLI Phases

Octet RED 384 Supplies

User-Provided Required supplies:

  • ForteBio Dip and Read sensors
  • Black 96-well plate to soak sensors
  • Black 96-well or 384-well plate for samples and reagents
  • Pipettors for sample handling

Optional provided supplies and equipement:

  • Empty BLI Sensor trays to use as working tray during experiment
  • 8-sensor transfer tool
  • Refrigerated centrifuge with microplate rotor

BLItz Supplies

User-Provide Required supplies:

  • ForteBio Dip and Read sensors
  • 96-well plate to soak sensors

CMI-Provide Required Supplies:

  • Black 0.5 ml tubes (provided by CMI)

 

Popular ForteBio Dip and Read Biosensors for Kinetics* Part Number
Streptavidin (SA) biosensors 18-5019 (96/tray)
High Precision Streptavidin (SAX) biosensors 18-5117
Super-Streptavidin (SSA) biosensors (for small molecules) 18-5057
anti-His (HIS1K) biosensors 18-5120
Ni-NTA (NTA) biosensors 18-5101
Anti-Human IgG Fc biosensors 18-5010

* See ForteBio website for more sensor types

OCTET Black Microplates** Part Number
Greiner Bio-One 96-well black flat-bottom PP, 200 µL 655209 (VWR 82050-784)
Greiner Bio-One 384-well black flat-bottom PP, 80-120 µL 781209 (VWR 82051-318)
ForteBio 384-well black tilted-bottom PP, 60 µL 18-5080

** Only Greiner Bio-One brand, black microplates or ForteBio plates are recommended by ForteBio for the Octet sample and reagent plate.