I'm using MST to investigate protein-protein inteactions. Everything seems to be set up properly, no negative outcomes in the binding check. However, I keep on getting inconsistent traces from the same complex sample. No aggregates just different traces every time. I tried different incubation times at room temperature and on ice from 5min to 2h but nothing seems to resolve the issue. Sometimes the complex trace is above the target-only trace and sometimes below. In few cases the two traces are the same. What could be going wrong?
What do you mean with "complex sample"? Is the sample not pure or a mixture? Do you have buffer mismatches? Is the sample stable enough at increasing temperatures?
Your sample heats up significantly during MST, especially if you use higher MST powers. I was advised to use low MST power and use only the temperature jump after 1.5 - 2.5 s for analysis if the signal-to-noise ratio allows that. If you then determine the Kd at later timepoints or higher MST powers and the Kd changes, probably your sample changes.
I think it is both the buffer and the MST power. Today I tried lowering the power and changed the buffer. The results are more consistent but now I have to get rid of the aggregates. Any ideas other than BSA and change of pH would be useful.
For most biophysical techniques you need highly pure and stable protein to obtain reliable results. All protein preparations that I use to determine affinities were purified by SEC, then checked with SDS-PAGE and high resolution mass for size, and by DLS for aggregation. If the preparation passes all of these tests, I typically don't have to worry about its behavior in the assay. In your case it is difficult to judge where to start improving the assay, because all of these parameters are unclear from the information you provide. It could be that you purified a protein complex and this is not stable in the buffer you use, that's why it aggregates. Or you concentrated it too high and it doesn't like that. Then the MST trouble shooting won't help because the problem is the protein preparation. I would make sure to have a high quality protein preparation and then follow the MST guidelines for troubleshooting (different capillaries, BSA, Tween-20 addition etc.). If this doesn't work, then use a different technique other than MST because for several systems it just doesn't work.
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