High purity and stability of your macromolecule are the hallmarks of successful samples in calorimetric experiments. The following procedures should be followed in sample preparation:
- All molecules should be gel or column purified before use.
- Macromolecules should be dialyzed against the buffer to ensure exact salt 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.
Chemicals to avoid
- DTT should not be used in ITC or DSC experiments. BME can be substituted instead.
- Tris buffer should not be used for DSC experiments. A buffer with a low temperature dependence of the pKa should be used instead.
Choice of buffer
Use buffer with ΔHion ~ 0 including phosphate, acetate, formate, citrate, sulfate, cacodylate, glycine.
Auto ITC concentrations and volume
Sample concentration in cell: ~10 * Kd
Concentration in syringe:
~ cell concentration * no. of binding sites * 10
- The easiest way to get a good starting concentration for a new system is to use the Experimental Design tab of the iTC 200 software.
Typical min and max concentrations: 3 mM to 500 mM
Sample cell volume ~ 400 microliters
Syringe volume ~ 120 microliters
DSC concentrations and volume
Typical min and max sample concentration: 0.1mg/mL to 10 mg/mL
Sample cell Volume ~ 400 microliters
Buffer ~ 200 milliliters