My protein stays in solution in 250mM imidazole, 5mM MgSO4 and 5% glycerol. However, I'm trying to make a simpler buffer to reduce the 'noise' in ITC analysis. I'm wondering on using 2.5% glycerol or 5% for my ITC buffer. previously I made a buffer of 100mM imidazole and 50mM NaCl and my protein crashed so, I'm thinking of making a 200mM imidazole and 2.5% glycerol instead.
Roughly what are you titrating? And at which pH is this experiment conducted? The glycerol itself is fine for ITC; the 'noise' could easily result from buffer ionization events. As such, I'd try retaining the MgSO4 but decreasing the imidazole -- can you use an inorganic salt instead? You don't need that much imidazole for buffering, but you could need it to make the solution salty enough. Finally, the best tip for ITC is to make sure your target and titrant are both dialyzed against the same buffer, whatever it is. (Hopefully you're already doing that, but it's worth mentioning.)
As Ryan said, glycerol is fine for ITC. I have done titrations with up to 25% glycerol, so 5% glycerol should work. Of course, you must have glycerol in both cell and syringe solutions.
Noise must be due to other reasons (aggregates, bending of the syringe,...), but not due to glycerol. Buffer ionization events should not contribute to noise either, but to heat background (usually called "heat of dilution").
You could employ MST (MicroScale Thermophoresis). It's a fast, easy and precise method to quantify biomolecular interactions. And the nice thing about MST: you will get feedback regarding sample quality and properties. For example, you will see immediately if your sample is aggregating thus enabling you to perform straight-forward assay/buffer optimization.
Since MST requires only tiny amounts of sample, you could optimize the conditions easily without wasting too much sample. And then you could run an ITC experiment with these optimized conditions in case you would like to verify your result with an orthogonal, independent method.
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