Sample Preparation

General Tips

  • The high purity and stability of sample preparation is critical for successful calorimetric and spectroscopic measurements.
  • Samples must always be centrifuged immediately before analysis.

Specific Tips

Circular dichroism

  • Sample amount and concentration – Proteins, DNA or organic molecules should be measured from 5-50uM (far UV) or 50-200uM (near UV), 50uL to 2mL.Your protein solution should be at A280 < 1.0 in the running buffer.
  • Buffer – Phosphate buffer at 20-50 mM is recommended; Hepes and Tris buffer may absorb at 190 – 200 nm and should be avoided. If possible, avoid salt, detergents and additives, if not, low concentrations of salt and additives recommended are: EDTA <1 mM; Salts <50 mM.

Critical reagents like DTT, β-Mercaptoethanol, Imidazole and DMSO absorb light and must not be present in the sample.

 

Fluorescence

  • Before starting your experiment, take a spectrum of your sample and your buffer solution (blank) to check absorptions and establish the wavelength that will be used.
  • Check all the calibrations of the device.
  • For fluorescence intensity experiments, bring the same solution as your sample to blank your measurement.
  • The amount and concentration of your samples will be dependent on the fluorophore’s quantity in your sample.

UV-Vis spectra

  • Avoid using high salt concentrations, additives, detergents or β-Mercaptoethanol; if necessary, use low concentrations.
  • If your solution is very concentrate, dilute it to keep your experiments into Lambert-Beer conditions.

Isothermal Titration Calorimetry

  • All molecules should be column purified by gel filtration before use.
  • Protein and ligand solutions need to be in the same buffer, to avoid heat of dilution in the measurement. All the samples must be dialyzed against the buffer to ensure the exact salt and additives amount matching in the reservoir and syringe samples. This dialysis buffer should be used as the reference in the experiments and any small molecule ligands should be dissolved into the dialysis flow-through.
  • Molar concentrations of the protein, RNA, or small molecule should be accurately known, preferably by absorbance measurements, to obtain the best quantitative results.
  • The macromolecule should be stable under experimental conditions. Precipitation will make the data unusable and potentially may damage the instruments. It is strongly suggested that sample stability be tested before proposal submission, by incubating a small fraction of the sample, at the experimental temperature for 1 h, and checking for precipitation.
  • DTT and TCEP should be avoided in your sample, BME can be substituted instead.
  • Sample concentration in cell: ~ 10 times the expected Kd, Concentration in syringe: ~ 10 times the cell concentration, multiplied by the number of binding sites. If you do not have any idea about your Kd, a good start might be 50uM in the cell and 500uM in the syringe.
  • Sample cell volume ~ 2000 uL, syringe volume ~ 300 uL
  • Use buffers with ΔHionization ~ 0, like phosphate, acetate, formate, citrate, sulfate, cacodylate, glycine. Quaternary amines (e.g. Tris) have high ΔHionization and should be avoided.
  • As a control, it is mandatory to perform the titration of the syringe material into buffer, and the peaks should be constant and similar in magnitude to those at the end of the sample titration experiment.
  • Solutions need to be degassed for 10-15 minutes before performing the experiment to avoid bubbles interference with heat transfer.

 

Differential Scanning Calorimetry

  • A typical sample concentration for DSC is 1 mg/ml, a range of 0.1—2 mg/ml may be used. To measure aggregation or other events, initial concentration should start around 2mg/ml and be decreased in additional measurements.
  • Some buffer components (e.g., DTT or metals) can have their own heat transitions which will be measured by the calorimeter and may interfere in your result, when it is possible avoid these components.
  • Protein and ligand solutions need to be in the same buffer, so no heat of dilution will be generated when they mix. The macromolecules should be dialyzed against the buffer to ensure exact salt and additives matching of the sample. This dialysis buffer should be used as the reference in the experiments and any small molecule ligands should be dissolved into the dialysis flow-through.
  • Sample cell Volume ~ 700 μl, Buffer ~ 700 uL
  • Solutions need to be degassed for 10-15 minutes before performing the experiment to avoid bubbles interference with heat transfer.

 

 Dynamic light scattering

  • A protein concentration of 0.5 – 0.75 mg/ml is a good starting range. For smaller proteins, better readings are obtained with higher concentrations.
  • Spurious particles must be removed from your sample. A 10-15 minutes centrifugation is recommended, since filtration can produce shear forces and binding problems. If that’s not feasible, spin filters work well for small volumes.
  • Load your sample into the cuvette with a gel-loading tip to avoid air bubbles. For the 12ul cuvette, load 20ul and for the 45ul cuvette, load 55ul. Put the cap on the cuvette so dust does not enter your sample.

 

Analytical ultracentrifugation

  • For a Sedimentation Velocity run, samples should present absorbance from 0.2 to 1.2 units. For OD>3, use the Sedimentation Equilibrium centerpieces (small volume). To decrease nonideality, keep protein concentration <1 mg/ml.
  • Use buffer of >50 mM ionic strength to suppress the nonideality stemming from charge effects. It is important to use a non-absorbing buffer.
  • DTT should be avoided as its spectroscopic properties change over time, TCEP is preferable.
  • Glycerol should be avoided due to viscosity, which may cause back diffusion.
  • Dialysis the sample against its buffer overnight. Save some of the dialysis buffer to put into the reference cells.
  • For a velocity cell, it is necessary 200-450 μL of each sample and buffer, respectively. Each cell stands for one sample. For equilibrium cells, it is necessary 120-150 μl of sample and buffer respectively, for each pair of sectors.
  • The experiment should be performed between 4°C and 25°C, according to the protein stability. For a run at 4°C it will be necessary to chill both the centrifuge and the rotor overnight first, which means that one day of the turn scheduled will be spent with equipment setup.
  • For Sedimentation Velocity runs, 42000 RPM is suitable for small globular proteins. For large proteins, 30000 RPM will do. For Sedimentation Equilibrium runs, start slowly and get to equilibrium there, then increase two steps, waiting for equilibrium in each time. There is a table in the Beckman manual (p. 4-26 – Support Material) to help you select rotor speeds for equilibrium runs, based on your monomer’s MW.