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Reichert SPR Systems: FAQ

Can SPR detect conformational changes?

Yes! The 2SPR system has an extremely high sensitivity, allowing it to detect very minor conformational change. We have even managed to detect cis-trans conformational changes in small organic compounds, ~360 Da.

Can SPR determine binding kinetics?

Quite definitely yes – SPR is the gold standard for determining binding and/or adsorption kinetics. Thanks to recent advances, the latest generation of SPR instruments are able to determine binding kinetics of molecules smaller than 100 Da. This is significantly better than other techniques such as surface-acoustic-wave technology (SAW) or QCM (quartz-crystal-microbalances), which currently lack to sensitivity to provide kinetic data on the interaction of small molecular entities and their corresponding targets.

Can SPR calculate binding affinity?

Certainly! The sensitive instrumentation and easy-to-use software allow the binding affinity of any particular ligand to be calculated. As a general comment, however, we would recommend that calculation of binding kinetics also be performed, due to the increased information provided on the interaction process. Another advantage of binding kinetics calculations is that they are not limited by the measurement technique used, being applicable in ITC, MST (microscale thermophoresis), etc.

Determining the concentration of biological compounds (e.g. antibodies) in crude samples

SPR is commonly used to determine the concentration of a range of biomolecules, including proteins, peptides, nucleic acids, and more. Using target-specific chip coatings allows these assays to be performed in complex mixtures, including serum and cell lysates.

How can I determine the stoichiometry of my interaction?

When using SPR, the signal change observed when progressing to equilibrium and saturation is proportional to the mass of the analyte bound to the chip surface. Provided the molecular weights of the ligand and analyte are known (even roughly), it is possible to calculate the binding stoichiometry.

Is it possible to measure very weak interactions?

Yes, by combining the sensitivity and high data acquisition rates of current SPR instruments with high ligand-capacity surfaces developed by XanTec. Using this powerful combination, even very weak binding events with fast on- and off-rates (e.g. carbohydrate-carbohydrate interactions) can be measured.

Is it possible to use pure organic solvents, or concentrated acid/base solutions?

The fluidics system of the SR7000DC and 2SPR instruments is extremely flexible. While they are predominantly used under bio-compatible conditions, the fluidic/chip system is easily able to perform accurate measurements in most organic solvents, as well as in the presence of concentrated acid/base.

Can I follow photosensitive or photo-triggered reactions?

Yes! XanTec offers quartz-window flow cells, which allow directed light-passage into the fluidics system. Because of this, it is possible to perform SPR measurements on photo-switching systems or photo-induced reactions.

Is it possible to combine SPR with large-scale particles? (whole cells, viruses, bacteria or nano-particles)

Certainly! While more optimal methods for particulate analysis exist, our SPR systems are designed with flexibility in mind. As such, XanTec carries a range of flow-cells and cuvettes which are suitable for studies with particulate ligands/analytes. If you are overwhelmed by the choice, simply ask us for recommendations based on our long experience.

Does SPR work with membrane-associated proteins?

Membrane-associated proteins are a challenge for many analytical systems, but are easily examined using SPR. By reconstituting proteins into nanodiscs, liposomes, vesicles or lipid bilayers, they can be directly bound to a suitable chip surface. From there the analyte interaction can be easily measured in a number of different assay formats, e.g. kinetic titration (single-cycle kinetics ®) experiments.

Is DMSO a problem during SPR measurements?

No. DMSO can alter the strength of the signal received by changing the bulk refractive index of the solution. However this is easily corrected by comparison to the reference channel, calculation of excluded volume correction values, and measurement of a calibration series containing equivalent concentrations of DMSO.

How crucial is the immobilisation step in SPR measurements?

As SPR involves examining the binding of analytes to a surface, successfully immobilizing the ligand is obviously important. Significant development has occurred in this area, and so a number of immobilisation techniques are available to choose from. Next to the 'traditional' covalent coupling (in which the ligand is randomly immobilized across the sensor surface), it is also possible to direct immobilization via a variety of commonly used tags (e.g. His6-tags, Strep-tags, Rho-tags, etc.) to achieve higher levels of "active" protein for binding.

In some rare cases users have reported problems with the ligand immobilization stage of their SPR experiments. However, when we investigated further (as part of our ongoing tech-support for customers), we found that over 90% of these cases were due to a failure to follow protocol steps: forgetting to remove free amines from the immobilisation buffer; using slightly high salt concentrations or incorrect pH; having impurities within the buffer solutions; or accidentally using inactive ligands.

Does SPR exhibit non-specific interactions?

Most SPR hydrogel chip surfaces utilize carboxymethyldextran to create the 3-dimensional matrix. This material can occasionally be problematic, leading to non-specific binding, cross-reactivity or ligand inactivation. To avoid this XanTec has pioneered the development of new hydrogel materials such as the linear polycarboxylate HC family, which show fewer non-specific interactions and better signal-to-noise ratios. We have then progressed even further by developing the HLC family of hydrogels, which prevent the majority of non-specific interactions by virtue of their extremely low electrostatic surface charge.

Unfortunately the same cannot be said for techniques utilizing chromophore/fluorophore interaction, such as microscale thermophoresis techniques (MST). In this technique, one interacting compound is commonly modified with fluorescent dyes – this adds a number of new hydrophobic regions which can in turn significantly increase non-specific binding. This is a particular problem when working with small molecular entities.

Are there any limitations regarding buffer compositions?

No! Provided the proteins being examined are not negatively influenced by the buffer or its additives, any buffer composition is possible. XanTec instruments are even able to provide accurate measurements in solutions containing high urea concentrations, strong acid/bases, or even organic solvents.

How can I verify the quality of my protein during the run?

There are several simple tests which can be used to check the quality of ligands and analytes. Ligand activity is usually checked by injecting the analyte at a concentration approximately 10-times the estimated KD value. Once binding has reached saturation, the ratio of molecular weight to bound/immobilized response units (µRIU) provides a good overview about the ligand activity.

What are the benefits of a modular system?

Unlike the 'black-box' approach followed by some of our competitors, the modular concept used in our equipment allows you to easily upgrade and maintain your system. Additional modules provide significant flexibility in experimentation, while easy access to all parts of the system allows customers to do much of the maintenance themselves if they desire – though XanTec is of course available to provide any assistance needed.

Can the sensor chips be coated with thin films (spin-coating or electro-spinning)?

Yes. As part of our commitment to flexibility, sensor chips are delivered for use without chip holders. As such they can be coated offline with any surface coating technique desired (e.g. electrodeposition, sputtering, dip-, spray- and spin-coating etc.).

I need to perform SPR experiments at temperatures > 40°C...

Not a problem! The 2SPR / SR7000DC come with Peltier heating elements as a standard, which allows heating of the system to temperatures of up to 70°C. Higher temperatures (up to and, if necessary, beyond 95 °C) are also possible with the purchase of specialized modules.

Do the instruments have an integrated µ-fluidic cartridge (IFC)?

No! Integrated fluidic cartridges, which are used in Biacore systems, are both extremely expensive (over €8000) and prone to errors as they age and clog over time. Our modular system has been designed from the bottom up to promote flexibility of design.

All valves, connectors and tubing used in our SPR systems are derived from robust and durable HPLC systems. Well-known and long-established manufacturers provide our pumps, syringes and autosampler, and thus guarantee that spare parts will be available for the life of the machine – a long time indeed! Additionally, each flow cell sold by XanTec uses state-of-the-art manufacturing techniques, employing extremely precise CNC machines to form flow cells from high-performance, biocompatible polymer resins.

What is the result? A flow channel volume under 100 nano-litres, with the durability to allow cleaning via sonication in detergent or organic solvents. We have sold these systems for many years, world-wide, and in that time have found that the fluidics are remarkably durable and long-lasting.

What consumables do I need?

We know that the modern laboratory has equipment from many different sources, and so designed our system to be as flexible as possible. The autosampler is compatible with standard HPLC vials, well-plates and their associated sealing methods – there is no need for proprietary flasks and caps for sample storage! This philosophy is continued with the capillaries and fittings of the SPR system, which have been selected from standardised FPLC components.

To complement our range of instrument hardware, XanTec offers the widest selection of SPR sensor chips around – with surface chemistries that can cover practically every application. Our experts are happy to assist you to select the optimal sensor chip surface. We can even develop a tailor-made surface for your more difficult experiments.

What are the yearly maintenance costs?

This is, naturally, dependent on how often the instrument is used. But what if we take a normal level of use (2-3 experiments per week) and assume that the operators are following the correct storage/standby guidelines? In this case, maintenance costs will be between €300-700 per year. This is surprisingly low, thanks to the modular replacements possible with XanTec systems, a similar Biacore set-up would be around €10.000.

How much does an experiment cost?

Let's look at an example kinetic determination experiment, determining the kon and koff values of the interaction between two unlabeled proteins. A standard sensor chip using carboxy-methylated dextran will cost between €65-75 from XanTec (a Biacore equivalent is notably more expensive, at €142-200). The initial immobilisation steps mean that the very first experiment with a new chip will take 1-2 days, and then 0.5 days for every subsequent experiment.

How much protein would I need?

This depends on many factors, but we will look at a typical kinetic experiment. The amount of ligand required (the immobilized species) will range between 2.5µg (for larger proteins such as antibodies) and 25µg (for smaller peptides and DNA). The analyte needed varies between 1-20 µg for medium/high affinity binding (pM-nM) and 20-500 µg for low affinity binding partners, or fragments (µM-mM). In a kinetic experiment targeting, say, antibody binding to a cancer antigen, 100 µL of a 10 µM (1.5 µg/ml) stock solution is sufficient.

How can XanTec systems save me time and money?

Does it save time? Yes, definitely. Using the Autosampler module allows the analysis of large numbers of samples independent of human interaction, allowing employees to do other things with their precious time. The speed of analysis provides results in a fraction of the time that an ELISA would take, while containing far more information in binding parameters.

Now let's examine costs. XanTec systems are designed to be modular, so you can easily purchase the system you need to have, then upgrade later as the lab grows. This modular design also makes maintenance and repairs quick and cheap. Our flow cells are accurate, good value and durable; unlike the expensive integrated fluidics systems used by competitors.

Can I easily transfer my data?

XanTec systems are designed to work within the modern, networked laboratory. The data, graphs and charts of your runs can be easily exported into a number of popular formats (including Clamp, Scrubber, TraceDrawer, image files, and .txt) for use in further analysis – this allows you to easily share your methods with outsourcing partners and collaborators.

What value can XanTec systems add to my workflow?

Modern biotechnology is built upon reliability and rapidity. Cell lines need to be validated, tumour-directed antibody affinity needs to be verified, and regulatory bodies usually require monitoring of a range of attributes throughout the manufacturing process. Automated systems such as XanTec SPR allow high-throughput, label-free monitoring of a wide range of concentrations, kinetics, and profiling data. The reproducible, quantitative data allows you to optimize many stages of a development or production workflow – while rapid results allow for iterative approaches to biomolecule design, reducing development time and increasing efficiency.