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> Molecular Interaction Measurements by Analytical Ultracentrifugation
> Molecular Interaction Measurements by Surface Plasmon Resonance

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Measurement of binding between two molecules is important for the understanding how these molecules interact with one another. The Hartwell Center for Bioinformatics and Biotechnology has a Biacore 3000 for measuring the interaction between two molecules. This instrument uses an optical phenomenon called surface plasmon resonance coupled with a microfluidic delivery system to detect binding of one molecule in solution with another molecule immobilized to a surface.

Surface plasmon resonance is an effect observed when polarized light is shined on a suitable surface material such as gold. The angle of the reflected light is affected not only by the surface but also by the composition of the medium very near the surface. As the refractive index of the medium near the surface changes, the reflectance angle changes. The Biacore instrument exploits this effect to measure the amount of material (protein, DNA, etc.) bound to the surface. If one of the molecules of interest is bound to the surface (termed the ligand) and the second molecule of interest is flowed over the surface (termed the analyte) the concentration of the analyte molecule near the surface increases as it binds to the ligand. The increase in concentration results in a change in the refractive index that the instrument detects as a change in reflectance angle. The change in this angle is proportional to the amount of analyte bound to the ligand. Since these measurements can be made very rapidly, real time binding data can be obtained which can provide information about the kinetics of the binding reaction.

The Biacore instrument uses a sensor chip to immobilize the ligand in a flow cell. A variety of immobilization methods are available to accommodate different types of molecules. The immobilization of the ligand is the most critical step in the process. The ligand must be attached to the surface without affecting its ability to bind the analyte. Since the flow cell, with its bound analyte, will be used for repeated binding measurements, the attachment must be stable to conditions that will remove bound analyte without affecting the binding ability of the ligand. For proteins, two immobilization methods are available. The protein can be coupled chemically directly to a sensor chip that contains a carboxymethyl dextran surface either through attachment to amines or to sulfhydryl side chains. The direct attachment method provides a stable attachment to the surface and in many cases does not affect analyte binding. In some cases, the ligand may be attached in more than one orientation, resulting in a mixture of binding kinetics.

Another method is to permanently attach a molecule that will bind the ligand such as an antibody. The capturing molecule can be an antibody specific to the ligand or can bind to a region introduced to the ligand for this purpose such as a GST fusion protein or a polyhistidine region. The ligand is then bound to the capturing molecule (antibody) in a single orientation than with the direct coupling method. This method will require more of the ligand to obtain a set of binding data since the regeneration of the surface between binding experiments will remove the ligand from the capture molecule and will need to be replaced for each binding experiment.

Methods are also available for attachment of DNA, membranes or liposomes, or even whole cells to the surface. Once a stable sensor chip surface has been produced and methods of regenerating the surface between binding experiments have been developed, the binding data can be obtained for any number of analytes. The Biacore 3000 system is equipped with an autosampler for automated injection and data collection. An example of the data generated in one such cycle is shown below.

There is an initial increase in the resonance signal corresponding to binding of the analyte to the ligand. The rate of this increase is a function of the concentration of the analyte and the association rate constant, k_on. After the injection of the analytes is complete, the system switches back to flowing buffer through the flow cell. At this point, there is a gradual decrease in the resonance signal. This decrease in signal results from the release of bound analyte from the ligand and is a function of the rate dissociation rate constant, k_off. From these two values, the equilibrium dissociation constant K,D can be obtained as K_D=k_off/k_on. Measurements of concentration are also possible since the height of the resonance response change is proportional to the concentration of the bound analyte and therefore proportional to the total concentration of the analyte in the solution. The last phase of the cycle is to regenerate the surface by removing the bound analyte and preparing for the next binding measurement. Depending on the molecules involved and the attachment method used, up to 100 binding experiments can be done in a single flow cell.

Each sensor chip has 4 flow cells. In order to control for any nonspecific binding effects, these flow cells are usually used in pairs with one flowcell containing bound ligand and the other without ligand used as a control. The analyte is flowed over these two surfaces sequentially, first through the control cell then the cell containing the bound ligand. Difference sensograms are obtained for the specific binding by subtracting the response in the control cell from the response observed in the cell containing the ligand.

A number of different types of experiments are possible with the Biacore 3000. Some of the main types are described below, but other types may be possible depending on the information required. A list of references to specific applications is also available, references.

Prelim-Feasibility
Before any binding data can be collected several preliminary studies are required to determine whether the experiment can be accomplished successfully.

1. Conditions must be identified for immobilization without altering or destroying the binding properties of the molecule.
2. Appropriate binding conditions for binding of the analyte to the ligand must be identified.
3. Conditions for regeneration of the sensor chip without altering or destroying the binding properties of the ligand must be identified. A set of feasibility experiments should always be conducted first to determine if these conditions can be met.

Kinetics
The Biacore 3000 is able to obtain over a wide range of binding rates.
The typical ranges for these constants are:
k_a= 10³-10⁷ M^-1s^-1
k_d= 10^-1-5X10^-6 s^-1
K_D= 10^-4-10^-11 M

A typical kinetics experiment requires injections of several different concentrations of analyte and should best be done in duplicate or triplicate. As many as 30 injections may be required to obtain good kinetic data. Since as many as 100 binding measurements can be performed in a flowcell, the kinetic constants of several analytes can be determined in one pair of flow cells. The data for a set of kinetic measurements would look like the figure below. The software is able to fit the data to a range of binding models depending on the conditions of the experiment.

Affinity
If either k_on or k_off is outside the measurable range of the instrument, an equilibrium method for obtaining the affinity constant, K_D, is possible. To obtain a reliable equilibrium constant, as with kinetic measurements, a number of measurements are needed at different analyte concentrations.

Affinity Ranking
For some systems, the actual rate or equilibrium constants are not as important as the relative affinity of a number of analytes for a single ligand. In these cases, relative binding strength can be determined and approximate affinity constants can be calculated from one or two binding measurements at one analyte concentration. This type of measurement may be most useful when only a small amount of the analyte is available or when a large number of analytes are to be compared.

Binding
It is also possible that binding affinity may not be needed and the question is simply does the analyte bind to the ligand? A single measurement at one analyte concentration is sufficient to answer this question.

Concentration
The concentration of active analyte in a solution can be measured with the Biacore 3000. A standard curve covering the concentration range of the samples is required. This standard curve must be constructed from the specific molecule being measured since the specific binding of the analyte to the ligand is being measured. This type of measurement may be useful for example to measure the concentration of a specific antigen in serum samples.

Ligand Fishing
The specific interaction of the analyte with the ligand allows the analyte to be detected even if it is not in a pure form. If a solution containing a mixture of analytes flows over a sensor surface with a ligand immobilized to it, the analytes, which bind to the ligand, will be attached to the surface of the flow cell. The Biacore 3000 is capable of recovering the bound analytes in a small volume. The recovered analytes can then be subjected to other analysis methods to identify the analytes, which bound to the ligand. This process termed ligand fishing, can be useful for identifying molecules which bind to the ligand.

Sample Requirements
The ligand must be pure. The recommended standard of purity is that it should be at least 90% pure on a silver-stained gel. If the ligand is to be immobilized through capture with an antibody as described above, it can be somewhat less pure since the specificity of the capture amounts to an additional purification step. The analyte need not be pure but must not contain contaminants, which would bind to the ligand or to any capture molecules, which may be used for immobilization.

Buffer selection is important in these experiments.
The preferred buffer is
10mM HEPES, pH 7.4
150 mM NaCl
3 mM EDTA
0.005% polysorbate 20

Since this buffer was designed to minimize nonspecific binding to the sensor chip and the microfluidics system. Other buffers can be used if required. The main consideration in choosing a buffer is that it must not inhibit the specific binding being measured. Another important consideration is refractive index of the buffer. The refractive index of the analyte solution should be the same as the system buffer. This condition can usually be met by dialyzing the analyte solution against the system buffer. Buffers with a very high refractive index (i.e. high salt) should be avoided since they produce a high background level that can decrease the dynamic range of the system. Very low ionic strength buffer solutions should be avoided since these solutions often lead to high levels of nonspecific binding to the flowcells.

Samples should not be radioactive or contain any biohazardous materials.

How to ORDER

1. Consult with Molecular Interactions staff concerning experimental design and sample requirements.
2. Print out the Biacore Request Form in a word doc or pdf format.
3. Prepare samples as discussed in step 1.
4. Submit samples and request form to the Molecular Interactions section in room D1011.
5. You will be notified by E-mail or telephone when the analysis is completed.
6. Schedule a meeting to discuss the results of the analysis.

Quality Assurance

1. Preventative maintenance is performed on the instrument on a regular basis to insure that it is in good working order.
2. All samples include a reference cell control which controls for binding to components in the system other than the specific interaction being measured.
3. All runs include a blank control which corrects for baseline drift and injection artifacts.
   In most experiments replicate samples are used to eliminate insure reproducibility of the results

Data Analysis/Troubleshooting

You will be notified by E-mail or telephone when the analysis is complete. At that time you should schedule a meeting with the Molecular Interactions staff to discuss the results. During the meeting, the method of analysis and the controls used in the analysis will be described. The results of the experiment will be provided and any problems will be described. A hard copy of the final analyzed data and a written summary of the experiment will be provided. Suggestions of how to improve the data or further experiments which could provide more information will be discussed.

Instrumentation

Molecular Interaction Measurements by
Surface Plasmon Resonance -Biacore 300

Fees

Please follow this link to a general fees page.