BIAcore Facility

BIACORE TECHNOLOGY: BINDING MEASUREMENTS, LABEL-FREE, IN REAL TIME

Overview

A Biacore 3000 instrument is available for your use in a core facility maintained by the Department of Physiology. Biacore sensor technology provides a versatile, highly sensitive, easy-to-use, and quantitative approach to study the kinetics and affinity of binding interactions under exquisitely controlled conditions. The technology relies on “surface plasmon resonance”, small changes in the interaction of monochromatic light with a metallic surface that occur when a protein or other molecule binds to that surface. Many different kinds of binding reactions can be studied. Extensive sets of binding curves can be obtained robotically. User-friendly software is available for curve-fitting and extraction of kinetic constants. Many different surface chemistries are available that are compatible with a wide range of ligands and binding reactions. Ligands can be proteins, nucleic acids, lipids, or carbohydrates. The size of the ligand is usually not limiting, and the technology allows all measurements to be made “label-free”.

The Biacore 3000 housed in Core Facility (Rm. 435 Howard Hall) is accessible to faculty, students, fellows and staff members. Training in its use can be obtained from the Core Operator, Dr. Y. Zhang (x 6-4410; x 6-2036; yzhan004@umaryland.edu). User fees, assessed for training and for machine time, are low by national standards; supplies are provided at cost. For more information, please contact Dr. Zhang or the Core Director, Dr. R. Bloch (x 6-3020; rbloch@umaryland.edu).

Surface Plasmon Resonance

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.

For more information on Biacore technology and SPR, please see http://www.biacore.com/lifesciences/index.html  

Special Features of the BIAcore 3000:

• (i) 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.
• (ii) 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.
• (iii) 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.
• (iv) 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.
• (v) 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.
• (vi) 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).

Location, Access, Fees and Contacts

The Biacore 3000 is housed in the Biosensor Core Facility, located in Rm 435, on the 4th floor of Howard Hall, a central location within the School of Medicine and on the University of Maryland, Baltimore, campus.

If you want to learn to use the instrument, you should obtain hands-on training, offered by the Biacore group in New Brunswick, N.J., several times a year. Inquire at this website.

Alternatively, 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.

To learn more about the services offered by the Biacore facility and its staff, please contact the Core Operator, Dr. Yinghua Zhang (6-4410; yzhan004@umaryland.edu) or the Core Director, Dr. Robert Bloch (x6-3020, rbloch@umaryland.edu).