Biacore T200 is a versatile, label-free system for detailed studies of biomolecular interactions. The system delivers high quality kinetic, affinity, concentration,specificity, selectivity, comparability, and thermodynamicinteraction data – in real time with high sensitivity. It is fully automated and enables fast screening of many interactions. The chips are equipped with four flow cells offering three ligands to be tested simultaneously (first flow cell serves as a reference cell for non-specific binding). For the high throughput analysis microtiter plates with 384 wells can be used. Analysis temperature range spans from 4° C to 45° C and temperature control of sample cmpartment is possible for the unstable samples. Built-in buffer selector enables up to four different buffers to be tested in the same run. Sample recovery for identification by mass spectrometry is possible.
|Sample type||from LMW drug candidates to high molecularweight proteins (also DNA, RNA, polysaccharides, lipids, cells, and viruses)|
|Flow rate||1-100 μl/min|
|Typical concentrations (proteins)||pM-μM|
|Sample volume||2-350 μl|
|Analysis temperature||4-45 °C|
|different buffer conditions||yes|
|Sample concentration||10-3-10-11 M|
|Association constant (ka)||103 to 3 x 109 M-1s-1 for proteins, 103 to 5 x 107 M-1s-1 for LMW molecules|
|Dissociation constant (kd)||10-5-1 s-1|
|Baseline noise||typically < 0.03 RU|
|Baseline drift||< 0.3 RU/min|
|Calibration-free concentration analysis||yes|
|Purity/quality of the sample needed||
Purity and homogeneity of analytes has to be exceptional. Proteins, for example, have to be tested with the SDS-PAGE with minimum of 1 μg protein loaded, where they should appear as a single well visible band (e. g. at least 95 % purity). Similarly, DNA has to be tested on agarose gel and lipid vesicles have to be tested with DLS for their homogeneity.
Concentration of the analyte needs to be determined as accurately as possible (spectrophotometrically) and it should be measured right before the experiment.
Purity of the ligand (molecule attached to the sensor chip) can be lower.
|Amount needed for a typical measurement||
Ligand (attached to the surface): ~ 10-50 µg of a protein/~0.1-0.3 µg of a DNA/~ 100–300 µl of 200 µM vesicles for one immobilization.
Analyte: ~200 µl of a biomolecule with a concentration around 10 x expected KD (per one titration).
Most commonly used buffers are HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005 % P-20), TBS-P (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.005 % P-20) and PBS-P (10.1 mM Na2PO4, 1.8 mM KH2PO4 pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.005 % P-20).
Additives to suppress non-specific binding are often added to buffers: 0.1 % BSA, 0.1–10 mg/ml CM-dextran (in case of dextran sensor chips), raising the detergent concentration (up to 0.1 %), raising the salt concentration (up to 250 mM NaCl), 3 mM EDTA.
When samples require glycerol or DMSO, special care should be taken to match ligand and reference surface.
Guest researchers are kindly asked to fill in the Sample request form regarding sample quality, experimental conditions and plausible previous results.
Buffers and consumables for experiments can be provided by the National Institute of Chemistry/Infrastructural Centre for Molecular Interactions Analysis by prior arrangement.