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Purpose

This protocol is used to determine the stability of your protein. A protein can be denatured either chemically, or thermally, (as here) and monitored using the circular dichroism spectrum. Typically, one wavelength of the spectrum (e.g. 222nm, for a helical protein) is chosen to monitor.

Equipment/Materials

• Jasco J-810 spectropolarimeter (Easton, MD)
• buffer (tris is not good for thermal melts due to pH dependence on temperature; phosphate is good, but not for CaBPs because it will precipitate with calcium ions; and note that Cl- absorbs at low wavelengths)
• protein, 200 microliters at around 2-50 uM
• enough LN₂ to keep mirror chamber evacuated
• 200 ul CD cuvette (not interchangeable with other cuvettes)
• the tiny stirbar (we have only one that's ideal for this; keep good track of it)

Steps

1. Review notes in binder by the Jasco for basic operation instructions.
2. Collect a spectrum of your protein to check for good (e.g. helical) character.
3. Collect baseline spectra of your buffer with any chelators, denaturants, etc.
4. To save yourself trouble, it may be worth collecting one spectrum at high temperature, just to ensure that you do see melting.
5. Use continuous spinning, and use Peltier thermostat (check water flow frequently at first, it tends to peter out.)
6. You're looking for a sigmoidal curve, showing little change at each end of the range.
7. You can plot data in Jasco Canvas, but be sure to export data as textfiles, as well, for external processing, later.

Parameters that worked for me:

• 222nm
• 5-100^oC
• 1^opitch
• 30 sec. delay
• 2^oC/min slope
• return to start (important to ensure reversibility)
• std sensitivity (100mdeg)
• resp: 4 sec
• 1nm bandwith

Variables

• denaturant concentration (if you have an ultra-stable protein which requires a constant level of denaturant to fully denature)
• salt concentration
• speed of thermal gradient 1-3^oC/min
• calcium or EDTA concentration for CaBPs

Last modified by Jonathan Sheehan

Alternative strategy: Chemical Denaturation

The advantage of doing a thermal rather than chemical denaturation is minimal sample-handling; our machine has automatic temperature control, but does not have a titration unit, so for a denaturant titration, repeated cycles of addition and mixing would be necessary. In some cases this may be preferable, however, and in those cases, note that the impurities in standard urea stocks make it unsuitible for CD analysis. Use ultrapure reagents (e.g. Fluka's urea #51456)