protein hplc question

[mtar]

Hi,
I would like to know if it is possible to separate protein dimer (through disulfide bridge and noncovalent interactions) from monomer in presence of DTT on C18 column? MW of monomer is ca. 14kDa, should I use urea or guanidine hydrochloride to overcome noncovalent interactions? Let me know if someone had tried something similar. Thanks.
MT

[HW Mueller]

Is DTT dithiothreitol?? Check out the chain started by CSV, "protein analysis" of Jan. 26?

[Mark Tracy]

The DTT+urea conditions will convert all the dimer to monomer and you will have nothing to separate. You probably will get better results with hydrophobic interaction chromatography. Protein separations on C18 are usually denaturing conditions, and that is probably not what you want.

HIC uses a weakly hydrophobic, large-pore stationary phase. The initial mobile phase is a strong salt like 2M ammonium sulfate that traps the protein on the column. A gradient to weak salt elutes the proteins. This method is good for preserving the biological and structural integrity of the proteins.

[Uwe Neue]

The best option for the separation of monomers from dimers is SEC. The mobile phase conditions can be made to be so mild that you can even separate non-covalently bound dimers from monomers.