Biophysical characterization - Instruct-CZ

Techniques are provided for the biophysical characterization of the structure, function and stability of biological macromolecules like proteins, nucleic acids, lipids and their complexes (Circular Dichroism, Differential Scanning Fluorescence, Differential Scanning Calorimetry, Dynamic Light Scattering, UV/Vis Spectrometry) and kinetic and thermodynamic parameters of their iteration (Microscale Thermophoresis, Surface Plasmon Resonance, Isothermal titration Calorimetry).

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User Guide

Circular dichroism spectrometer Chirascan Plus (Applied Photophysics)

Measurement of circular dichroism spectra and absorbance as function of temperature, pH and concentration 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. Detection range: 170-1150 nm. Peltier temperature control.

The CDNN software package is available for detailed model-based analysis and predicting secondary structure using CD data. Software Global Analysis of multiwavelength 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.

Not optimal for CD solutions, containing DTT, imidazole, glycerol, DMSO, high concentrations of salts.

Prometheus NT.48 (Nano Temper)

Measurement of protein stability of up to 48 samples at a time. No dye is required, tryptophan and tyrosine fluorescence at 330 nm and 350 nm is detected.

Temperature range: from 15°C to 95°C.

Sample concentration range: from 5 μg/ml to 250 mg/ml. At least 20 μl of a sample is required.

Protein must contain tryptophans or tyrosines in order to detect protein unfolding. For thermal unfolding experiments no assay development or special sample preparation is needed.

Microcal VP-capillary DSC differential scanning calorimeter (Malvern)

Measurement of 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.

Sample buffer and buffer in the reference cell should be exactly the same.

The sample solutions should be dialyzed 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.

If the presence of reducing agent is required for the sample, the use of up to 5 mM ß-mercaptoethanol or TCEP instead of DTT is recommended.

Since fluoride-containing samples cause irreparable damage to the VP-DSC cell, their use is prohibited.

Zetasizer Nano ZS90 (Malvern)

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.

25 μl of sample and the same volume of “empty“ buffer is needed. For protein solutions, 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.

UV/visible spectrophotometer Specord 50 Plus (Analytic Jena)

This device is a UV/Vis double-beam spectrophotometer with split-beam technology that combines high energy throughput with good stability. Temperature control with Peltier element, scan range 5 - 95ºC. Max wavelength - 190, min wavelength - 1100 nm. 50 - 1500 μl of sample.

Monolith NT.150 microscale thermophoresis (NanoTemper)

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.

Concentration of fluorescent labeled molecule is 10 to 100 nM. At least 20 μl samples per capillary is needed.

Final concentration of unlabeled molecule should be at least two orders of magnitude above the expected Kd value. To perform simulations of binding events and to help choose the appropriate concentration, the “Concentration Finder” software is available on the device control panel.

Monolith NT. LabelFree thermophoresis (NanoTemper)

Instrument uses intrinsic tryptophan fluorescence for microscale thermophoresis detection, thereby allowing label -free and immobilization-free experiments.

MST can be used fort he label and immobilization 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).

Concentration range of tryptophan-containing protein: 100 nM - 10 μM.

Final concentration of unlabeled 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 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 to screen analytes simultaneously against 36 different targets of interest, enabling rapid comparison amon glarge numbers of interactions. SPR can be used for: 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.

Concentration of ligand depends on the level of immobilization 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.

Microcal iTC200 isothermal titration calorimeter (Malvern)

Instrument is used for the characterization 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 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.).

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 ionization 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.