NIC: Sample requirements

Surface plasmon resonance (SPR) – Biacore X100

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/en/equipment/3/biacore-x100

Instrument specifications

Measurement possibilitiesaffinity (KD), kinetics (ka, kd), active concentration, thermodynamics
Sample loadautomatic
Size limit> 100 Da
Flow rate1–100 μl/min
Typical concentrations (proteins)pM–μM
Sample volume50–120 ml
Refractive index1.33–1.40
Analysis temperatureroom temperature
Number of flow cells2
Reference subtractionyes
different buffer conditionsyes
Sample concentration10-3–10-11 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 analysisyes
Single-cycle titrationyes

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 typeaffinity (KD), enthalpy (∆H), entropy (∆S), stoichiometry 
Sample volume2 ml
Cell volume1400 µL
Injection syringe volume300 µL
Sample capacity4–8 samples per 8 h
Noise0.5 ncal/s
Temperature range2–80° C
Response time20 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

MeasurementsAffinity (KD), enthalpy (∆H), stoichiometry, enzyme kinetics
Sample/experiment16
Fluoroscent chanells2 (red, green)
RangenM–mM
Labellingyes
Fluorescent molecule concentration10-9–­10-3
Complex media measurementsyes
Sample volume4 μl
Sample mass10 Da–104 kDa
Experiment and analysis timefew minutes
Immobilizationnot needed
Temperature range22–45 °C
Maintenancenot needed