Tips and tricks for preparing your samples for a successful experiment
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 clog the instruments.
Chemicals to avoid
- DTT should not be used - BME can be substituted instead. Ammonium cannot be used on Affinity ITC auto. Anything incompatible with sample cell. If in doubt, check with Julia or Neela.
- Affinity ITC auto - Gold
- MicroCal Auto-iTC200 - Hastelloy C-276 Alloy
Choice of buffer
Use a buffer with ΔHion ~ 0. Good choices include phosphate, acetate, formate, citrate, sulfate, cacodylate, and glycine.
Auto ITC concentration
The easiest way to get a good starting concentration for a new system is to use the Experimental Design tab of the Nano Analyse software.
- Sample concentration in cell: ~10 * Kd
- Concentration in syringe: ~ cell concentration * no. of binding sites * 10
Affinity Auto ITC sample volume
Affinity ITC auto (TA Instruments)
- Sample cell volume – 485 uL minimum
- Syringe volume – 210 uL minimum (Can vary depending on number of injections/volume per injection)
MicroCal Auto-iTC200 sample volume
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 uM to 500 uM
- Sample cell volume: - 400 uL
- Syringe volume: - 120 uL
EXACT BUFFER MATCHING IS ESSENTIAL