detergents in gel filtration
nestgrp at world.std.com
Thu Jun 15 15:36:51 EST 1995
> On 7
> Jun 1995, P. Friedl wrote:
> > In article
<Jan.Eggermont-0206951120590001 at fysiologie-1.med.kuleuven.ac.be>,
Jan.Eggermont at med.kuleuven.ac.be (Jan Eggermont) says:
> > >
> > >Hello,
> > >
> > >I am currently investigating the oligomeric nature of a purified protein.
> > >On a Superose 12 FPLC colum with a 150 mM NaCl buffer the protein migrates
> > >with a Mr of around 100k whereas the predicted molecular mass from the
> > >amino acid sequence is 28k suggesting it may form a trimer. I now want to
> > >find out what type of interactions (hydrophobic/hydrophilic/S-S
> > >bridges...) are going on. I presume raising the salt concentration will
> > >unmask hydrophilic interactions and adding a reducing agent such as DTT
> > >will break up S-S bridges. However, I am not sure about the best way to
> > >tackle the hydrophobic interactions. At first I wanted to use Triton-X100
> > >but this will interfere with the UV recording at 280 nm. In addition I was
> > >told that because of the low critical micellar concentration (cmc) of
> > >Tx100, some of the protein may be incorporated into micelles. So any ideas
> > >out there on a detergent that does not absorb at 280 nm and that has a
> > >rather high cmc?
> > >thanks in advance.
> > >
If you add organic solvents, EtOH, MeOH, PrOH, or MeCN you can mask the
hydrophobic components. If at the same time you raise the salt conc. to
ca. 250 mM you should have more than enough to overcome the hydrophilic
interactions. You could probably get away with the salt at 100 mM.
Andrews and Alpert published a mixture of 25% MeCN with 250 mM sodium
sulfate 5 mM KPO4 pH 3 for the HYDROXETHYL Aspartamide SEC columns but
that was to force all the proteins in the standard mixture to behave in a
SEC mode alone. See also Eur. J. Biochem 202, 589-595 (1991) for their
mix on this column.
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