By Mike on Tuesday, March 30, 2004 - 08:33 am:
I have a population of a specific protein that is misfolded (disulfides) and a population that is folded correctly. On a C18 column (ACN, TFA), I can distinquish 2 distinct peaks, but not baseline resolved. Is there another way to better resolve these two protein isomers, from others experience, that I can collect these peaks for further analysis?
By tom on Tuesday, March 30, 2004 - 12:06 pm:
Reversed-phase is, almost by definition, a denaturing technique. If you expect the native forms of the proteins to be more different from each another than the denatured forms, then a non-denaturing technique (HIC or ion-exchange) might give you better selectivity.
If you don't mind the fact that you would be collecting denatured protein,
- and if your resolution on the C18 column is close,
- and if you're not intending to collect a lot of the protein(s),
- and if run time isn't a big issue (a lot of ifs!),
then the path of least resistance may simply be to couple two C18 columns columns together: cut the flow in half to keep the same pressure and quadruple the gradient time to keep the same selectivity and you should increase your resolution by about 40%.
Other possibilities:
If the misfolding gives the two proteins significantly different shapes (different hydrodynamic radii), then GFC might do the trick.
If the misfolding changes the geometry of an "active site", then affinity chromatography using a ligand specific for that site should give you very high selectivity.
Let us know what you try (and how it works!) so that we can all learn from your experience!
By Anonymous on Tuesday, March 30, 2004 - 07:35 pm:
In your protocol, shouldn't one get double the resolution?
By tom on Wednesday, March 31, 2004 - 09:58 am:
No, that's a common misconception.
Doubling the column length will (approximately) double the plate number, but resolution is a function of the *square root* of the plate number. The resolution increase is therefore about 40% (the square root of 2 = 1.4).
By Anonymous on Wednesday, March 31, 2004 - 03:39 pm:
But you are not only doubling the column length, you are also reducing the flow rate, which - for a large molecule, means that you are doubling the plate count again.
By Uwe Neue on Wednesday, March 31, 2004 - 04:42 pm:
There are other ways in which you can increase resolution, but I need to know more about your LC conditions. My suggestion would be to drastically flatten the gradient in the range where the two peaks elute. Of course, this is only of value if the rest of the chromatogram is of no interest to you.