Sample Preparation

General Tips

  • High purity and stability of your macromolecule are the hallmarks of successful samples in calorimetric and spectroscopic experiments. All proteins used must first be centrifuged.
  • Always refer to a responsible in case of doubt or problems with the equipment.
  • It is not allowed alter the voltage or any other specification of equipment and is not allowed to take any equipment from the plug.
  • After finishing the experiment, turn off the equipment when no other user is ready to use.
  • At the end of the experiment, leave the cuvette properly washed, dried, and deliver it to the technician.
  • Remember that the computers are used exclusively for managing appliances and some data analysis. Not for browse internet and check emails. The data storage is responsibility of the user and if remains in the computer can be deleted without notice.

Specific Tips

Circular dichroism

  • Proteins, DNA or organic molecules, from 5-50uM (far UV) or 50-200uM (near UV), 50uL to 2mL.
  • Your protein solution should be at A280 < 1.0 in an appropriate buffer.
  • The buffers should be able to appropriate measurements. It is recommended the use of phosphate buffer at 20-50 mM, Hepes is not recommended because it absorbs at 195nm. If possible avoid salt and additives, if it is not, low concentrations of salt and additives are recommended like: 1-20 mM, EDTA: <1 mM; Salts: <50 mM; Lack of detergents.
  • Critical reagents: DTT, β-Mercaptoethanol, Imidazole and DMSO absorb light and must not be present in the sample subjected to experiments of circular dichroism spectroscopy.

Fluorescence

  • Try to use the most pure solution.
  • Before to start you experiment take a spectrum of your sample and your blank solution, in order to check possible absorptions of the sample, and to check the wavelength that will be used.
  • Check all calibrations of the device
  • Try to have the more information about your system and probes
  • For fluorescence intensity experiments, bring the same solution as your sample in order to blank your measurement.

UV-Vis spectra

  • Try to use the most pure solution
  • Avoid using high salt concentrations or additives like detergents and β-mercaptoethanol, if necessary to keep the stability of your solution, use low concentrations.
  • If your solution is very concentrate, dilute it, keep your experiments into Lamber Beer conditions.

Isothermal Titration Calorimetery

  • All molecules should be gel or column purified before use.
  • 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.
  • 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 could potentially damage the instruments. It is strongly suggested that you test this before submission by incubating a small fraction of your sample at the experimental temperature for 1 hr and checking for precipitation.
  • DTT and TCEP should be avoided in your sample, BME can be substituted instead.
  • Sample concentration in cell: ~ 10 * Kd, Concentration in syringe: ~ cell concentration * no. of binding sites * 10. If you do not have any idea about your Kd, a good guess could be 50uM in the cell and 500uM in the seringe.
  • Typical min and max concentrations: 3 mM to 500 mM
  • Sample cell volume ~ 2000 uL, syringe volume ~ 300 uL
  • Use buffers with ΔHionization ~ 0, Including 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 so that bubbles don’t interfere with heat transfer

Differential Scanning Calorimetry

  • A typical protein concentration for DSC work is 1 mg/ml through a range of 0.1—2 mg/ml can be used. To look for aggregation or other events, you can start at 2mg/ml and then decrease concentration. When deciding what buffer to use, keep in mind that some components (e.g., DTT or metals) can have their own heat transitions which the calorimeter will pick up.
  • 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.
  • If diluting from stocks be very careful use a balance rather than pipettes
  • Centrifuge or filter sample if there is a possibility of aggregated material, but beware that aggressive filtering can denature proteins
  • Adopt and stick to a standard reproducible protocol
  • Sample cell Volume ~ 700 μl, Buffer ~ 700 uL Tris buffer should not be used for DSC experiments. A buffer with a low temperature dependence of the pKa should be used instead.
  • DTT should not be used in ITC or DSC experiments. BME can be substituted instead.
  • Solutions need to be degassed for 10-15 minutes before performing the experiment so that bubbles don’t interfere 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. You need to remove spurious particles from your sample so they don’t scatter light. A 10-15 minute spin in a microfuge is recommended, since filtration can produce shear forces and binding problems. If that’s not feasible, spin filters work well for small volumes if you pre-wet them with buffer and spin that through first.
  • Load your sample into the cuvette with a gel-loading tip so you don’t introduce 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. Give the cuvette several sharp taps on the log book to dislodge any microbubbles.

Analytical ultracentrifugation

  • For a Sedimentation Velocity run, it is necessary enough protein to be seen by absorbance (0.2—1.2A units for linear response). For OD>3, use the Sedimentation Equilibrium centerpieces. 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.
  • Dialyzis the sample against its buffer overnight (with 3 changes) so that the chemical potential of the macromolecule is defined under conditions of constant chemical potential of solvent. Save some of the dialysis buffer to put into the reference sectors.
  • For a velocity cell, it is necessary 200-450 μL each of 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. Each cell stands for three samples.
  • 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 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.