MoB-IBT: Sample requirements

Microscale thermophoresis (MST) Monolith NT.115 instrument (Nano Temper)

This is an immobilization-free technique for the measurement of biomolecular interactions with nM-mM range of affinities.

Typical sample requirements:

  • Target (the fluorescent molecule) ~ 200 µl of labeled target per one titration. Concentration of unlabeled protein to use with a labeling kit: 100 μl of 200 nM (His-tag dye), 100 μl of 2-20 µM (amino-reactive and maleimide-reactive dyes).
  • Ligand (the non-fluorescent binding partner) ~ 20 µl per titration at the concentration starting at a 100-fold above the expected Kd, but not below 100 nM.

Other appropriate requirements:

  • For the protein labelling commercially available labelling kits are used (amine, Cys or His-tag reactive).
  • Standard buffer recommendation – 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 good 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. This is to avoid primary amines in the buffer competing with the labelling reaction.
  • DTT and β-mercaptoethanol interfere with the labelling reaction and therefore need to be avoided.

Microscale label free thermophoresis Monolith NT.LabelFree instrument (Nano Temper)

uses intrinsic tryptophan fluorescence for microscale thermophoresis detection

Typical sample requirements:

  • Target ~ 250 μl of 0.5-1 μM protein with intrinsic protein fluorescence.
  • Ligand ~ 50 μl of non-fluorescent binding partner at least 20 x above Kd.

Other appropriate requirements:

  • One of the binding partners needs to have at least one tryptophan, while the second binding partner must not show autofluorescence at the same wavelength.

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

For monitoring and quantification of interaction of biomolecules.

Typical sample requirements:

  • 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;
  • Immobilization of one interacting partner is essential; sensor chips with linker chemistry are commercially available.

Other appropriate requirements:

  • The acetate buffer (at pH 4.0, 4.5, 5.0, or 5.5) is recommended as immobilization buffer. The recommended running buffer for most applications is the phosphate buffered saline, pH 7.4 (10 mM sodium phosphate and 150 mM sodium chloride with 0.005% Tween 20).

Isothermal titration calorimetry MicroCal iTC200 (Malvern Panalytical)

Instrument for the characterization of biomolecular interactions of small molecules, proteins, antibodies, nucleic acids, lipids etc.

Typical sample requirements:

  • Concentration of protein in a sample cell should be at least 10-fold higher than the expected Kd.
  • Ligand concentration in syringe should be 20-fold higher than protein in the cell – 50-600 μM (for 1:1 stoichiometry). Ligand volume for the titration syringe: 120 μl (syringe volume 40 μl; for sample injection and control injection into buffer).

Other appropriate requirements:

  • Both macromolecule and ligand must be in identical buffers to minimize heats of dilution. The buffers used should have low ionization enthalpies (e.g. phosphate, citrate, acetate).
  • The pH of component solutions must be matched to avoid additional heat from pH equilibration.
  • If a ligand needs DMSO for solubility, the same final DMSO concentration should be in both binding partners. The recommended upper limit DMSO is 10% for binding studies.
  • Reducing agents TCEP and β-mercaptoethanol are recommended over DTT at concentration up to 5 mM.
  • The recommended upper limit of glycerol is below 20% (v/v).

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

Technique for the determination of thermodynamic parameters of macromolecule unfolding that results from heat-induced denaturation.

Typical sample requirements:

  • Typical protein concentrations 0.2 – 2.0 mg/ml. Concentration of oligonucleotides 80-300 µM.
  • Sample volume: 250 μl

Other appropriate requirements:

  • 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.
  • Buffer should have a low enthalpy of ionization (e.g. glycine, sodium/potassium acetate, sodium/potassium phosphate, sodium cacodylate, sodium citrate). PIPES, HEPES and MES should be avoided.
  • Since fluoride-containing samples cause irreparable damage to the VP-DSC cell, their use is prohibited.

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

Instrument is used for the determination of secondary structure of proteins and conformation of oligonucleotides.

Typical sample requirements:

  • Measurement of CD spectrum for the determination of secondary structure of protein requires 160 µl of 0.1 – 0.2 mg/ml protein solution.
  • Measurement of CD spectrum for the determination of DNA conformation requires 160 µl of 20 µM solution.

Other appropriate requirements:

  • Phosphate buffers with little to no NaCl are recommended.
  • Solutions, containing DTT, imidazole, glycerol, DMSO or high concentrations of salts are not optimal for CD.

Differential scanning fluorescence (DSF) Prometheus NT.48 (Nano Temper)

Technique for the protein melting temperature determination.

Typical sample requirement:

  • Concentrations of protein should be from 5 µg/ml to 150 mg/ml.
  • Sample volume needed is 10 µl.

Other appropriate requirements:

  • Proteins should contain tryptophan and tyrosine. No dye is required, tryptophan fluorescence at 330 nm and 350 nm is detected.

Dynamic light scattering (DLS) Zetasizer Nano ZS90 and Zetasizer Ultra (Malvern Panalytical)

Instruments for the measurement of particle and molecular size, with the option of measuring zeta potential and electrophoretic mobility, and molecular weight using static light scattering.

Typical sample requirements:

  • Minimum concentration of protein is 0.1 mg/ml.
  • Sample volume is 30 μl.
  • It measures particle and molecule size, from 1 nm to 10 µm.

In-plate DLS SpectroLight 610 (Xtal-Concepts)

Instrument is equipped with white light and UV imaging capabilities. It measures particle radius in a range of 1 nm to 5 µm and enables imaging in visible and UV light (crystallization monitoring).

Typical sample requirements:

  • Minimal drop volume is 0.2 μl.
  • Minimum concentration of protein is 1 mg/ml.

Other appropriate requirements:

  • The instrument measures in drops located in SBS-format crystallization plates, or Terasaki plates.

Photoluminescence Spectrometry FLS1000 (Edinburgh Instruments)

Photoluminescence spectrometer with ns time resolution.

Typical sample requirements:

  • Highly diluted solution of photoactive compound is required. Sample concentration is usually in nM – μM range and highly depends on molar extinction coefficient.
  • Sample volume is 50 μl – 2 ml.

Fourier-transformed Infrared spectrometry (FTIR) spectrometer Vertex 70v (Bruker)

With time resolution. FTIR spectra analysis can be used for the estimation of protein secondary-structural components in aqueous (both H2O and D2O) solutions.

Typical sample requirements:

  • Small amounts of high-purity (>95%) proteins at concentrations >0.5 mg/ml.
  • Sample volume is 20-50 μl.

Additional accessories:

  • Spectra collection can be done in transmission mode (Liquid Cell) or in reflection mode (ATR – attenuated total reflection). Samples for different modes should meet some specific requirements.
  • Transmission mode/Liquid cell: Solvent may highly interfere with sample spectra, so D20 solvent is highly recommended for protein structure measurements. Otherwise, protein concentration should be increased to 3 mg/ml and higher. Any ammonium salt or urea must be avoided.
  • Bio-ATR cell: Protein can be dissolved in water at minimum concentration 20 mg/ml. FTIR spectra of sample in solid state can be collected as well.

Correlative optical tweezers – fluorescence microscopy C-Trap (Lumicks)

Instrument enables simultaneous manipulation and visualization of single-molecule interactions in real time.

Typical sample requirements:

  • The instrument is especially suited for (but not restricted to) studies of protein interactions with nucleic acids or cytoskeletal filaments, conformations of nucleic acids, protein folding and conformational changes or liquid-liquid phase separation. The sample requirements depend to a large extent on the experimental design.

Small angle X-ray scattering SAXSpoint 2.0 (Anton Paar)

A state-of-the-art laboratory instrument with high flux X-ray source (MetalJet, Excillum) and highly sensitive detector (Eiger, Dectris). The technique allows high level characterization of particles in solution and assessment of sample quality. High X-ray flux and autosampler allow high throughput screening experiments. Both measurement cell and autosampler are temperature controlled.

Additional accessories:

  • UV-Vis absorption spectrometer (CaryUV 60, Agilent) – in situ sample monitoring.
  • Liquid chromatography system (ÄKTA go, GE Healthcare) – last minute sample separation; operable inline or offline with SAXS.

Typical sample requirement:

  • Minimal volume: 10 μl.
  • Sample concentration: 1-10 mg/ml; typically, concentration series are advised.

Other appropriate requirements:

  • Blank buffer has to be exactly the same with the sample buffer; (dialysis suggested as the last step of sample preparation).

Glovebox (Glove Box)

Rigid chamber (155 x 85 x 70 cm) equipped with a binocular microscope, double-door antechamber and an oxygen level meter, which can be filled with nitrogen.

Pipetting robots NT8 (Formulatrix), Gryphon (Art Robbins Instruments), OT-2 (Opentrons)

  • Automatic pipetting robots able to work with small volumes (down to 10 nl for NT8). All robots work with standard SBS format plates. NT8 and Gryphon are used for dispensing microvolumes (typically, but not only, crystallization drops). OT-2 is more versatile and programmable for various experimental purposes, but with higher minimal volume.