Protein/Ligand Preparation
The key to a successful ITC run is dependent on pure samples. Aim for 99%+ purity using dialysis just before ITC and ensure the protein and ligand are in identical buffers. The slightest contamination or buffer mismatch (especially a difference in pH) will show up as heat signatures which will confuse the interpretation of your data.
If the Kd value is unknown
If you know nothing of the expected association (Ka) or dissociation (Kd) constants then us
280ul of 5mM protein in sample cell and
40ul of 50mM ligand in syringe.
in identical buffers.
The syringe : cell molar ratio is approx 10:1 so scale it if you can't afford that much protein or ligand. From your result you might be able to get a ballpark Kd figure.
If you have an idea of Kd values, then calculate the optimal protein concentration
Let's get some definitions out the way:
Ka = Association constant, expressed as M^-1
Kd = Dissociation constant, expressed as M
[P] = protein concentration in the cell in M
[L] = ligand concentration in the cell in M
n = number of binding sites
To get a good binding curve showing good initial heats and proceeding to saturation, we need to ensure the "c" value is within range, which is an expression of confidence in being able to derive binding parameters from a given set of data.
A c value between 5 and 500 is necessary, the optimum range being between 10-100 ideally.
c = n.[p].Ka
So your protein concentration should be between
[P]=100/n.Ka
[P]= 10/n.Ka
Your ligand concentration will be about
[L]=n.10.[P]
Cleaning ITC cell and syringe
The ITC cell is made of Hastelloy C276. There are strict chemical compatibility guidelines but in general no strong acids, bases or organic solvents.
Run a water-water sample to ensure any peaks are 0.05 ucal/sec or less. Anything more may indicate a manual water wash of the cell and syringe is required (10 times with milliQ water should do it).
Only if you really need to use Decon wash solution but this should be reserved for heavy precipitation or lipids inside the cell - most of the time this will be overkill. Do not run decon through the auto-washing unit - rinse out the decon from the cell a few times before plugging in the auto washer.
A good water-water run is critical before starting your experiment, so don't skimp on this!
Loading Samples
Ensure your samples are at room temperature (not fresh from ice) to avoid temperature differences affecting your results.
Degas them, filter them to avoid precipitates and bubbles if possible.
When loading the cell do so without introducing foam - if you do that, you'll probably need to rinse the whole cell and start again because removing foam is extremely difficult by the normal method.
The run
Watch the reference power carefully to ensure it is what it should be. If it's not accurate, you'll need to restart.
At the beginning of a run, if your injections are too small or the spacing between injections is insufficient, feel free to modify your injection protocol for all the remaining injections. The software will calculate the correct delta H values even if every injection volume or spacing is different so that at least you can get some result (not ideal but better than throwing the sample out).
Finishing the run
Clean out with water as soon as possible to prevent proteins crystallizing or precipitating in the equipment.
When you are done for the day, please remember to refill the reference and sample cells with MilliQ water.