BIACORE TECHNOLOGY: BINDING MEASUREMENTS, LABEL-FREE, IN REAL TIME
Quantitative measurements of binding of one ligand to another is an essential step in understanding how macromolecules interact with each other under physiological conditions. Many interactions between macromolecules are now studied in qualitative ways (e.g., yeast two-hybrid screen, gel or blot overlays, cotransfections followed by immunofluorescence or immunoprecipitation), but none of these procedures provides information about affinity or kinetics of binding. Without this information, it is difficult to be sure that the interactions one detects using such procedures actually occur in a living cell.
BIAcore biosensor technology is a versatile, highly sensitive, label-free, and easy-to-use approach to study binding interactions quantitatively, under exquisitely controlled conditions. The technology relies on the phenomenon of “surface plasmon resonance”, small changes in the reflection of monochromatic light from a metallic chip that occur when the chip’s surface binds a protein or other molecule. The nature of the bound molecule and the “receptor” for that molecule that has been attached to the metallic surface are not critical. As a result, BIAcore technology has been used effectively to study protein-protein, protein-oligonucleotide, protein-carbohydrate, and oligonucleotide-oligonucleotide interactions. The BIAcore facility, which currently houses a BIAcore 3000 instrument, is available for use on a fee-for-service basis by the university community, and by researchers at other universities and biotechnology companies.
Surface Plasmon Resonance
Location, Access, Fees and Contacts
Location: Biosensor Core Facility, Rm 435, on the 4th floor of Howard Hall
Hands-on training: Biacore group in New Brunswick, N.J.
Fees: The facility can run your samples for you. Current rates for academic investigators at the University of Maryland, Baltimore, are $35/hr, if you run the samples yourself, or $65/hr, if your samples are run by facility staff. Higher rates ($125/hr) apply to investigators at other institutions.
Surface plasmon resonance (SPR) occurs when light strikes the surface of a thin metal film at an angle that should permit total internal reflection of the incident light. As the evanescent light wave passes through the film and reflects from the opposite surface, which is in contact with a liquid medium, it excites “surface plasmons”, clouds of electrons that behave as single particles. These generate an electrical field that extends ~100 nm into the medium. Resonance conditions vary with the wavelength and angle of incidence of the light, and with the nature of the medium that interacts with the surface plasmons. Any change in the medium in the vicinity of the foil surface can alter the wavelength and angle of incidence of the light that is capable of causing SPR.
Such changes are largely independent of the chemistry of the molecules involved. Thus, anything introduced into the medium that binds to the sensor surface can alter the SPR signal. A major advantage of SPR is therefore that it can be used to detect a wide range of molecules, including proteins and peptides, carbohydrates, and nucleic acids, without the need to introduce labels of any type.
The Biacore 3000 uses a light-emitting diode as the source of monochromatic light, gold as the thin metal film, and a set of optical detectors to measure the change in the angle of the light reflected from the foil-medium interface. (All other light is filtered out). This angle is linearly related to the amount of material bound to the sensor surface of the gold film that is exposed to the medium. Any change in the SPR signal, e.g., a change associated with the association or dissociation of a molecule to the surface, is recorded against time and stored digitally. The recorded signal is expressed as “Resonance Units”, or “RU”. In the Biacore 3000, an RU is equivalent to the binding of 1 pg of protein per mm2 of the sensor surface, and a signal as small as 2 RU can be reliably detected.
Find out more about Biacore technology and SPR.
Special Features of the BIAcore 3000:
- It is sensitive. The Biacore 3000 can detect clear signals over low backgrounds in binding studies of molecules as small as maltose (360 Da). More recent models, which we are now testing, are even more sensitive and reliable in studies of low molecular weight ligands.
- Many different surface chemistries are available from Biacore, allowing a wide variety of bimolecular binding reactions to be analyzed. The most frequently used Biacore sensor chip, the CM5, is constructed with a thin layer of dextran on the surface of the gold foil exposed to the medium. The dextran can be readily derivatized to produce surfaces containing a variety of generally useful reagents, such as streptavidin (e.g., the SA sensor chip) and antibodies. Metal chelation with the NTA chip and hydrophobic derivatives, made with the HPA chip, are also available. In addition, Biacore manufactures a series of unmodified or altered chemical surfaces in the “Pioneer Chip” series. All of these chips can be used multiple times, reducing the cost per assay.
- The Biacore 3000 has 4 independently monitored channels. This effectively creates 4 potentially identical biosensor surfaces that can either be used independently, in pairs, or all together. The same or different ligands can be immobilized on each of the sensor surfaces, and binding to each can be compared simultaneously to analyze binding kinetics or specificity. This reduces the noise of non-specific binding, allows for correction of artifacts due to the flow of solution over the biosensor, and increases the accuracy of measurement or antibodies to fusion protein partners, such as glutathione-S-transferase (GST) or epitope tags.
- The hydraulic system minimizes the volume of samples used and recovery of samples is easy. The Biacore uses an injection loop like that of HPLC units. The dead volume is small (< 2 µl in multichannel mode), and volumes as small as 5 µl/channel can be analyzed.
- The unit is easy to program and to operate robotically, permitting unattended use for extended periods of time. Runs can be programmed overnight or even over a weekend.
- Curve-fitting is easy. Biacore software facilitates “global curve-fitting”, permitting the determination of kinetic and binding constants from a set of binding curves generated with different concentrations of analyte in a single set of calculations. Software features, termed “Wizards”, make data collection and analysis easy. More advanced software permits the extraction of kinetic and binding constants under conditions in which mass transport effects may be significant (see below).