The temperature range for the DSC is 1° to 110°.
The temperature range for the Thermo-Vac is 2° to 80°. To tighten its bleeder valve properly, first screw down the upper knob, then the lower one.
VP in the instrument name stands for Valerian Plotnikov, the father of microcalorimetry technology.
Peaks going up are endothermic.
A peptide which is all random coil won't show a transition even at 5 mg/ml because there isn't enough structure. You'll only see a mild deflection of the baseline.
Phosphate buffer sometimes makes for noisy DSC traces. Acetate and HEPES are okay. Tris is not recommended since its pH changes with temperature. (NaHCO3 too?) Keep the metal content of your buffer low.
DTT will act at a certain temperature and change your protein's conformation. Lots of cysteines in your protein could be a problem at high temperature because they find each other. Do your scans without DTT first. (See the Reducing Agents document for further details and recommendations.)
The pH of your buffer can affect whether you see a peak or not. Samples up to pH 12 can be run.
Since proteins aggregate maximally at their pI, work away from the isoelectric point and lower the concentration as much as possible to prevent aggregation.
5% glycerol in your buffer will make filling trickier bubble-wise. Just move slowly.
To collect both upscans and downscans, make all the upscans the odd-numbered ones by choosing Unique Scans, Odd, then entering the starting and final temps., etc. Follow a similar procedure for making the downscans the even ones. Scan rate maxima are 90°/hr. for upscans and 60°/hr. for downscans.
If you ask for only upscans (from 10° – 65°) and let it cool itself back down (rather than do a downscan), it takes only 26 min. to get back down.
If you're using downscan data, you should have a 5 min prescan time to help get better data. The thermal core can achieve a stable temperature before scanning begins.
Buffer/buffer scans to set thermal history:
You can have the first scan be a downscan by just setting the temperatures correctly in the DSC Control panel.
When adding more scans onto an existing set, the software will only duplicate scan 1 and not give you up and down scans. You always have to tell it to go back down.
Upscans and downscans won't overlay each other but all upscans and all downscans will overlay each other if everything's equilibrated.
If your buffer/buffer scans are parallel to each other but not lying on top of each other, more degassing is happening and the thermal capacity will be new after each degassing run. Eventually, the degassing will be finished and the curves will lie on top of each other.
Do not run buffer/buffer scans on a narrower temperature range than the one you'll use for your samples. You need the thermal history for the whole range.
DSC on lipids or DNA
Set a slow scan rate (20-30°/hr) so that hysteresis won't be an issue. Set the Filter Period to be 1-5 sec and the Feedback Mode to High. Don't start scans at <5°. (The DSC goes below the set point and freezing the cells destroys the instrument.)
A good concentration for DNA is 50 uM.
After putting lipid samples in the DSC, rinse cells three times with water, twice with isopropanol, then twice with chloroform, twice again with isopropanol, then three times with water. If protein was present, now wash with 5% Contrad detergent, then rinse with twice the normal volume of water.
The cells are not resistant to caustics or fluoride. They're fine with 10% bleach.
If protein aggregates in the sample cell due to being heated, cells need to be cleaned with
nitric acid. (Debby does this.) Aggregated protein really sticks to the walls.
If the pressure won't go up to ~30 psi, check that there's water in both cells. Low pressure and upscans/downscans saturating at -15/+15 indicate that one cell is empty or that the cap is not tight.
If a buffer/buffer scan gives a sudden straight-line trajectory (like an airplane taking off), the data is saturated indicating that the sample cell is much hotter than it should be. This is caused by either an empty cell or a bubble in the cell.
If buffer/buffer scans aren't overlaying or have a funny shape, wait until you get to the bottom of your temperature range. Loosen the cap, then tighten it again. This will release any bubbles out of the cell and then the baselines should overlay nicely.
If there were bacteria growing in a buffer, they would settle out over time and buffer/buffer scans would become reproducible.
If buffer/buffer upscans are more than 1-2 mCal/min away from zero, clean the cells.
To see the current scan overlaid on recently-collected ones, go to the Completed Scans screen and double click on Layer 1. Move over Data 1 (current scan). It will superimpose on the completed ones. Click on its name (upper right) to get a window for changing the color of the line.
It happened once that downscans were at +15 on the Completed Scans screen but when the data was loaded into Origin for analysis, it turned out they were only at +5.
After data has been stored, open Origin for data analysis but before you load your data, uncheck the side box for "scan rate normalize". This will display the data just as it was on the real time graph.
To remove an aberrant scan from a group, double click on that scan in the Data Analysis software. A window pops up which has a Remove button.
In the data analysis version of Origin, if the usual toolbar isn't at the bottom of the screen, go to View, Toolbars and choose Tools.
Do not do data analysis while the DSC is acquiring data, even just buffer/buffer scans. It will change them.