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

CMI OctetRED384 BLI Getting Started Guide

Bio-Layer Interferometry (BLI) Technology from Sartorius (formerly ForteBio)
Purchase BLI Dip and Read sensors from Sartorius (formerly ForteBio)
CMI OctetRED384 BLI Legacy Data Analysis Software Guide, for data analysis instructions using the older version of Data Analysis

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.