Surface plasmon resonance (SPR) – Biacore X100 and Biacore T200
Purity/quality of the sample needed
Purity and homogeneity of analytes has to be exceptional. Proteins, for example, have to be tested with SDS-PAGE with a minimum of 1 mg 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).
Buffer considerations
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 % P20).
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 working with lipid vesicles, buffers should not contain detergents.
Tris-containing buffers should be avoided in the amine-coupling (ligand immobilization) step. When samples require glycerol or DMSO, special care should be taken to match ligand and reference surface.
Other requirements
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. Additional information can be found at http://www.molekulske-interakcije.si/
Instrument specifications for Biacore X100
| Measurement possibilities | affinity (KD), kinetics (ka, kd), active concentration, thermodynamics |
| Sample load | automatic |
| Size limit | > 100 Da |
| Flow rate | 1–100 μl/min |
| Typical concentrations (proteins) | pM–μM |
| Sample volume | 50–120 ml |
| Refractive index | 1.33–1.40 |
| Analysis temperature | room temperature |
| Number of flow cells | 2 |
| Reference subtraction | yes |
| 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 |
| Single-cycle titration | yes |
Instrument specifications for Biacore T200
| Sample load | Automatic | |
| Sample type | From LMW drug candidates to high molecular weight 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 | |
| Refractive index | 1.33-1.40 | |
| Analysis temperature | 4-45C | |
| Flow cell | 4 | |
| Reference subtraction | Yes | |
| Different buffer conditions | Yes | |
| Sample concentration | 10-3-10-11 M | |
| Association constant (ka) | 103 to 3×109 M-1s-1 for proteins, 103 to 5×107 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 | |
| Single-cycle titration | Yes |
Isothermal titration calorimetry (ITC) – MicroCal VP-ITC
Purity/quality of the sample needed
Pure samples are required and concentrations determined as accurately as possible (spectrophotometrically).
Amount needed for a typical measurement
400 ml of the titrant (syringe) with a concentration approx. 20-fold higher than anticipated KD and 1.8 ml of the macromolecule (sample cell) with a concentration 10-15 ´ lower than the macromolecule concentration.
Buffer considerations
The two binding partners should be in identical buffers. DTT and unstable chemicals should not be used. DMSO should be matched extremely well between the cell and the syringe.
Other requirements
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. Additional information can be found at http://www.molekulske-interakcije.si/en/equipment/4/microcal-vp-itc.
Instrument specifications
| Measurement type | affinity (KD), enthalpy (∆H), entropy (∆S), stoichiometry |
| Sample volume | 2 ml |
| Cell volume | 1400 µL |
| Injection syringe volume | 300 µL |
| Sample capacity | 4–8 samples per 8 h |
| Noise | 0.5 ncal/s |
| Temperature range | 2–80° C |
| Response time | 20 s |
Microscale thermophoresis (MST) – Monolith NT.115
Purity/quality of the sample needed
Pure samples are required and concentrations determined as accurately as possible (spectrophotometrically).
Amount needed for a typical measurement
Target molecule can be fluorescently labelled on-site. Concentration of unlabelled protein to use with a labelling kit: 100 μl of 200 nM (His-tag dye), 100 μl of 2–20 µM (amino-reactive and maleimide-reactive dyes). Approx. 200 ml of the labelled target is needed per one titration together with 20 ml of the unlabelled ligand molecule with a concentration 100-fold above the expected KD (and not below 100 nM).
Buffer considerations
MST optimized buffer: 50 mM Tris pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.05 % Tween-20 or 0.1 % Pluronic F 127.
For the His-tag labelling it is advised to use PBS with 0.05 % Tween-20.
For the labelling with an amino-reactive dye, purified protein should be in a buffer that does not contain primary amines (e.g., ammonium ions, Tris, glycine, ethanolamine, glutathione) or imidazole. DTT and 2-mercaptoethanol interfere with the labelling reaction and therefore need to be avoided.
Other requirements
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. Additional information can be found at http://www.molekulske-interakcije.si/en/equipment/6/mst-monolith-nt115.
Instrument specifications
| Measurements | Affinity (KD), enthalpy (∆H), stoichiometry, enzyme kinetics |
| Sample/experiment | 16 |
| Fluoroscent chanells | 2 (red, green) |
| Range | nM–mM |
| Labelling | yes |
| Fluorescent molecule concentration | 10-9–10-3 |
| Complex media measurements | yes |
| Sample volume | 4 μl |
| Sample mass | 10 Da–104 kDa |
| Experiment and analysis time | few minutes |
| Immobilization | not needed |
| Temperature range | 22–45 °C |
| Maintenance | not needed |