These are guidelines suggested by MicroCal. For more information on DSC and its applications, check out the Malvern website.
- AVOID FLUORIDES COMPLETELY!! Fluorinated solutions will cause irreparable damage to the instrument.
- Since DSC experiments are performed over a broad temperature range, the pH of the buffer should have a low temperature dependence. This makes Tris a poor choice.
- To minimize artifactual heats, the 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 aren't as good but are used. For detailed information on numerous buffers see: J. Phys. Chem. Ref. Data (2002) 31:2 p.359.
- Choosing a pH a couple of units away from (usually below) the isoelectric point of the protein and keeping the ionic strength of the solvent low (10 to 50 mM) will help avoid aggregation after unfolding.
- Avoid using DTT as it can cause erratic baselines. If you must include a reducing agent, use β-mercaptoethanol or tris(2-carboxyl)phosphine (TCEP) at low concentrations (i.e. <5mM). TCEP is not stable in phosphate buffer.
- You will need ~1.2 mL of solution in order to fill the cell properly.
- Typical sample concentrations are 0.2-2.0 mg/mL. Higher concentrations will help your signal-to-noise, but may affect reversibility of the transition.
- You will need some buffer (>20 mL) for rinsing cells and running baseline controls.
Run Time Considerations:
- The sample should be dialyzed exhaustively against buffer to ensure that sample and reference solutions are exactly matched.
- Centrifuge sample 15-20min at 13,000 x g to remove particulates.
- If possible, the sample concentration and pH should be determined on the final solution. Accurate curve fitting requires accurate sample concentrations.
- Expect to do a minimum of 5 scans per DSC experiment.
- The first scan of an identical series is typically less repeatable due to a different 'thermal history' of the DSC prior to scan #1. The data are collected with buffer in both cells but are not used.
- Follow the initial 'dummy scan' with at least 2 more buffer/buffer scans to obtain an accurate baseline and assess reproducibility.
Once you are satisfied with the quality of your buffer/buffer scans, you can then proceed with running samples (see below).
- After establishing a reproducible buffer/buffer scan, you refill the sample cell while the instrument is cycling (w/o pausing or stopping the scans).
- There's typically a short time window for refilling (during the cooling period between scans), so plan appropriately to ensure that you don't miss the refill opportunity.
- Make sure that your sample is degassed and at the same temperature as the DSC cell, otherwise your baseline will be off.
- A repeat scan of the same sample is normally used to assess the reversibility of the transition. Recovery of >80% of the initial endotherm is usually considered reversible.
- Keep in mind that these experiments can take a while (a typical scan rate is 1 deg/min). You may want to set up buffer/buffer scans to run overnight, so you can begin running samples first thing the next morning.
- A pdf document on choosing DSC parameters can be downloaded here.