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About Molecular Biophysics, Vestec near Prague, Czech Republic

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Techniques are available for the analysis of kinetic and thermodynamic parameters of biomolecular interactions and for the biophysical characterisation of the structure, function and stability of biological biological macromolecules like proteins, nucleic acids, lipids and their complexes.

Instruments Available:

The Chirascan Plus CD spectropolarimeter with avalanche photodiode detector - provides fast scanning and high sensitivity. This instrument can simultaneously measure accurate CD, absorbance and fluorescence data. Detection range: 170-1150 nm. Peltier temperature control.

Instrument measures the temperature of thermally-induced structural transitions of molecules in solution. A complete thermodynamic profile is generated to understand the factors that affect conformation and stability of proteins, nucleic acids, micellar complexes and other macromolecular systems. The operating temperature range is of -10°C to 130°C. Maximum scan rates are 90°C/hr in the upscan mode and 60°C/hr in the downscan mode.

Instrument measure native DFS to determine protein thermal transition temperatures and stability of 48 up to samples at a time. No dye is required, tryptophan fluorescence at 330 nm and 350 nm is detected. Temperature range: from 15 °C to 95 °C.

Instrument is used for the measurement of particle and molecular size using Dynamic Light Scattering, with the option of measuring zeta potential and electrophoretic mobility, and molecular weight using Static Light Scattering. Size (diameter): from 0.3 nm to 5 microns. Molecular weight measurement down to 10 kDa. Temperature range 0-90°C.

Most advanced system for the measurement of particle and molecular size, particle charge and particle concentration. The measurement of particle concentration is calibration-free and suitable for a wide range of materials.It combines DLS and ELS system, incorporating Non-Invasive Back Scatter (NIBS) and Multi-Angle Dynamic Light Scattering (MADLS) technology for the measurement of particle and molecular size.

Direct measurement of submilimolar to nanomolar binding constants (103 - 109 M-1). Thermodynamic characterization of the molecular interaction in a single experiment (stoichiometry, Kd, ∆H and ∆S values). Calorimetric measurement over a range of biologically relevant conditions (temperature, salt, pH, etc.).

Characterization of protein-ligand interactions based on thermophoretic effect, using the intrinsic tryptophan fluorescence. No sample modification is required with this device

Biomolecular interactions that can be measured under close to native conditions based on thermophoretic effect. Protein labeling is required with this device.

Access to balances, pH meter, Nano-Drop, mixers, shakers, centrifuges, salts, buffers etc.

Protein interaction array system enables label-free quantitative analysis of biomolecular interactions in real time using SPR technology. The ProteOn system allows to screen analytes simultaneously against 36 different targets of interest, enabling rapid comparison among large numbers of interactions. Quantification of binding affinity and kinetics.; Determination of binding specificity and the number of binding sites. Characterization of membranes, lipids, nucleic acids and micellar systems.

Molecular absorption spectroscopy with ultraviolet and visible radiation in the spectral range from 190 to 1100 nm. Temperature control with Peltier element, scan-range 5-95ºC.

User Guide

Circular dichroism (CD) spectrometer Chirascan Plus (Applied Photophysics)

Measurement of circular dichroism spectra and absorbance as function of temperature, pH and concentration can be used to determine the secondary structure of proteins and peptides, conformation of RNA and DNA, as well as to detect conformational changes. Avalanche photodiode detector provides fast scanning and high sensitivity.

The CDNN software package is available for detailed model-based analysis and predicting secondary structure using CD data. Software Global Analysis of multi-wavelength kinetic data is available to fit multi-dimensional experimental data to one of a number of specified models. Measurement of CD spectrum for the determination of secondary structure of protein requires 160 μl of 0.1 - 0.2 mg/ml protein solution; for the determination of DNA conformation requires 160 μl of 20 μM of solution or 1400 μl of 2 μM solution. It is not optimal for CD solutions containing DTT, imidazole, glycerol, DMSO, high concentrations of salts.

Differential scanning calorimeter MicroCal VP-Capillary DSC (Malvern Panalytical)

The technique measures the temperature of thermally-induced structural transitions of molecules in solution. A complete thermodynamic profile is generated to understand the factors that affect conformation and stability of proteins, nucleic acids, micellar complexes and other macromolecular systems.

The sample buffer and buffer in the reference cell should be exactly the same. The sample solutions should be dialysed against the buffer solution used for the DSC measurements. Sample and reference cell volumes are 200 μl. Typical sample concentration: 0.2 - 2.0 mg/ml. 

The use of up to 5 mM ß-mercaptoethanol or TCEP instead of DTT is recommended if the presence of reducing agent is required for the sample. Use of fluoride-containing samples is prohibited because they cause irreparable damage to the VP-DSC cell.

Differential scanning fluorescense (DSF) assay Prometheus NT.48 (Nano Temper)

This technique allows the measurement of thermal shift assays of up to 48 samples at a time for examining binding interactions in proteins. Sample concentration range: from 5 μg/ml to 250 mg/ml. At least 20 μl of a sample is required. The protein must contain tryptophan or tyrosine in order to detect protein unfolding. No assay development or special sample preparation is needed for thermal unfolding experiments.

Dynamic light scattering (DLS) technique Zetasizer Nano ZS90 (Malvern Panalytical)

The instrument is used for the measurement of particle and molecular size using Dynamic Light Scattering, with the option of measuring zeta potential and electrophoretic mobility, and molecular weight using Static Light Scattering.

A 25 μl volume of the sample and the same volume of “empty“ buffer is needed. For protein solutions, a concentration of at least 0.2 mg/ml is required. For the measurements of zeta potential in folded capillary cells, 0.75 ml of sample is required.

Dynamic light scattering (DLS) technique  Zetasizer Ultra (Malvern Panalytical)

This is the most advanced system for the measurement of particle and molecular size, particle charge and particle concentration. The measurement of particle concentration is calibration-free and suitable for a wide range of materials. It combines DLS and ELS system, incorporating Non-Invasive Back Scatter (NIBS) and Multi-Angle Dynamic Light Scattering (MADLS) technology for the measurement of particle and molecular size.

Isothermal titration calorimeter MicroCal iTC200 (Malvern Panalytical)

This instrument is used for the characterisation of biomolecular interactions of small molecules, proteins, antibodies, nucleic acids, lipids etc. The iTC200 device can be used for: direct measurement of submilimolar to nanomolar binding constants (103 - 109 M-1); thermodynamic characterisation of the molecular interaction in a single experiment (stoichiometry, Kd, ∆H and ∆S values); calorimetric measurement over a range of biologically relevant conditions (temperature, salt,pH, etc.).

The volume of the sample placed in the cell must be at least 300 μl. Preferably, the solutions of macromolecules should be dialysed against the buffer solution used for the ITC measurement. The ligand solution (the sample placed in the injection syringe) must have a volume at least 50 μl. Normally, the ligand concentration should be 10 times as high as the concentration of macromolecule. In the case of high affinity interactions, the minimum concentration of macromolecule (that causes measurable heat effects) is 10 μM. 

For low affinity interactions the macromolecule sample concentration should be at least 5 times the Kd value. The buffer solution, containing both the macromolecule and the ligand of interest, should be the same. The buffers used should have low ionisation enthalpies (e.g. phosphate, citrate, acetate). If the presence of reducing agent is required for a protein stability, then ß-mercaptoethanol (at a concentration lower than 5 mM) or TCEP (lower than 2 mM) should be used rather than DTT.

Monolith microscale thermophoresis (MST) NT.115 (Nano Temper)

This MST technique allows to detect changes in hydration shell, charge or size of molecules and thus to detect biomolecular interactions. MST can be used for the determination using a fluorescent dye or fluorescent protein of the affinity of interaction from 1nM to mM. The concentration of fluorescent labelled molecule is 10 to 100 nM and at least 20 μl samples per capillary is needed. Final concentration of unlabelled molecule should be at least two orders of magnitude above the expected Kd value. The “Concentration Finder” software is available on the device control panel for performing simulations of binding events and for helping choose the appropriate concentration.

Monolith microscale label free thermophoresis NT.LabelFree (Nano Temper)

This instrument uses intrinsic tryptophan fluorescence for microscale thermophoresis detection, thereby allowing label -free and immobilisation-free experiments. MST can be used for the label and immobilisation free determination of protein binding to ions, nucleic acids, small molecules and sugars (with an affinity of interaction in the range of 10 nM to mM). The concentration range of tryptophan-containing protein is 100 nM - 10 μM. Final concentration of unlabelled molecule should be at least an order of magnitude or more above the expected Kd value. Molecular weight range: 10-107 Da. Minimum sample volume used: 10 μl per sample.

Surface Plasmon Resonance (SPR) system ProteOn XPR36 (BioRad)

The protein interaction array system enables label-free quantitative analysis of biomolecular interactions in real time using SPR technology. The ProteOn system allows screening of analytes simultaneously against 36 different targets of interest, enabling rapid comparison among large numbers of interactions. SPR can be used for: quantification of binding affinity and kinetics; determination of binding specificity and the number of binding sites; characterisation of membranes, lipids, nucleic acids and micellar systems.

Concentration of ligand depends on the level of immobilisation desired, generally 10-200 μg/ml. For kinetic analysis the best results are obtained by using a 100- fold range of analyte concentrations, 0.1-10 x Kd.

UV/Vis Spectrometer Specord 50 Plus  (Analytica Jena)

This device is a UV/Vis double-beam spectrophotometer with split-beam technology that combines high energy throughput with good stability. This device requires 50 - 1500 μl of sample.

Instruct Centre

BIOCEV

BIOCEV

Prumyslova 595

25250, Vestec, Prague

Czech Republic

www.biocev.eu

Molecular Biophysics, Vestec near Prague, Czech Republic

Contacts:

Tatsiana Charnavets
Tatsiana Charnavets
Institute of Biotechnology ASCR
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