Biacore X100 (GE Healthcare) is automated surface plasmon resonance based refractometer. It can be used for the analysis of 15 different analytes in single run. The chip with two flow cells are compatible with Biacore X. The unit has incorporated degasser and offers single-cycle titration and calibration-free concentration analysis.
|Measurement possibilities||affinity (KD), kinetics (ka, kd), active concentration, thermodynamics|
|Size limit||> 100 Da|
|Flow rate||1–100 μl/min|
|Typical concentrations (proteins)||pM–μM|
|Sample volume||50–120 μl|
|Analysis temperature||25 °C|
|Number of flow cells||2|
|different buffer conditions||yes|
|Sample concentration||10-3–10-11 M|
|Association constant (ka)||103–107 M-1 s-1|
|Dissociation constant (kd)||10-5–0.5 s-1|
|Baseline noise||< 0.1 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 by prior arrangement.