BIACORE
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BIACORE

BIA Technology

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BIACORE Technology

Overview:

Biomolecular interaction analysis (BIA) is a label-free technology that uses surface plasmon resonance (SPR) to monitor binding interactions between molecular partners.  The BIACORE integrates this technology with a microfluidic system to allow real-time monitoring of these binding interactions between molecules on a sensor surface.  The sensor surface is prepared by coupling one of the molecular partners to dextran, using standard chemistries such as amine and thiol coupling. This "surface" is actually a lawn of carboxymethyl dextran polymers to which the molecule of interest is attached, thus lending a certain degree of mobility to the bound molecule (ligand).  The partner molecule (analyte) is then passed over this prepared surface under flow, and any interaction between partners is recorded as a function of time in resonance units (RU) -- these are arbitrary units that reflect the changes in refractive index (a function of mass) at the sensor surface that occur as a result of the binding of solution-phase analyte to (or its release from) surface-bound ligand.

Applications:

The BIACORE can be used for addressing questions of:

SPECIFICITY:

Do my two molecules bind to each other?  If so, under what conditions?

CONCENTRATION:

How much of my protein is present?  What is my antibody titer?

AFFINITY:

How tight is the binding interaction?  What are the optimal conditions for interaction?

KINETICS:

How fast are the on- and off-rates for binding?  How stable is the complex?

MAPPING:

Does the epitope for antibody A overlap with that of antibody B?  Does my antibody block the interaction of my two proteins?

AVAILABLE CHIPS AND COUPLING CHEMISTRIES:
1) CM5 (Carboxymethyl dextran surface):

This chip is the most commonly used.  The following types of coupling chemistries are available for activating this surface:

AMINE.  Direct linkage via primary amines on the protein/peptide.  Surface activation is by EDC (N-ethyl-N'-(3-dimethyl aminopropyl)-carbodiimide hydrochloride), and NHS (N-hydroxysuccinimide).

SURFACE THIOL.  Direct linkage via thiols generated on the chip surface.  Surface activation is by EDC/NHS followed by preparation with Cystamine and DTT.

LIGAND THIOL.  Direct linkage via thiols on the protein/peptide.  Surface activation is by EDC/NHS followed by preparation with PDEA (pyridinyldithioethaneamine hydrochloride).

ALDEHYDE.  Direct linkage via carbohydrate on the protein/molecule.  Surface activation is by EDC/NHS followed by preparation with hydrazine.

2) HPA (Hydrophobic surface):
This chip is essentially a "blank" chip that lacks the carboxymethyl dextran of the CM5 chip.  The surface is composed of hydrophobic alkanethiol goups. This chip can be used to mimic conditions of an ELISA (hydrophobic adsorption of protein), or to lay down lipid monolayers of various compositions.  The latter can be used to study protein-lipid interactions or protein-protein interactions using a membrane-associated protein/receptor.

3) SA (Streptavidin surface):
This chip is a prepared CM5 chip with streptavidin coupled to the surface.  This surface can be used to "capture" any molecule that has been labeled with biotin (protein, peptide, DNA, oligos, etc.). 

4) NTA (Nickel Chelation surface):
This chip has been prepared with a nickel-chelating agent, and can thus be used to "capture" proteins/peptides containing His tags.

5) L1 (Hydrophobic surface):
This chip is useful for the rapid and reproducible capture of liposomes.

Dr. Padmaja Mehta-D'Souza