FPLC under Denaturing Conditions

mbarnhart at my-dejanews.com mbarnhart at my-dejanews.com
Thu Mar 4 11:04:41 EST 1999


Alejandro,

I did not make a mistake.  The particular resin with which the size exclusion
column was made had affinity for hydrophobic groups.  It even said so in the
descriptions.  At the time however, I was an undergraduate and no one in my
lab had any notion that a size exclusion column might have hydrophobic
affinity.

The issue is that denaturation exposed the hydrophobic groups of our protein
of interest, in this case RNAse T1.  When the protein was refolded (and it
refolds 100% or very close with RNAse T1), the hydrophobic groups associated
with each other and the protein came off the column.

This column was a Pharmacia product as was the FPLC, FYI.

Michael

In article <57C0B572D021D211834200A0C949D816114315 at MAILSERVER>,
  a.krimer at biosidus.com.ar (".Krimer Alejandro") wrote:
> Dear Michael:
>
> I think you have had some mistake. In general you can elute de proteins with
> water in Hydrophobic Interaction Chromatography, like Phenyl-Seph.
>
> Alejandro
> > -----Mensaje original-----
> > De:	mbarnhart at my-dejanews.com [SMTP:mbarnhart at my-dejanews.com]
> > Enviado el:	Wednesday, March 03, 1999 5:00 PM
> > Para:	methods at net.bio.net
> > Asunto:	Re: FPLC under Denaturing Conditions
> >
> > Hi,
> >
> > If you want to be certain of denaturation, you should go ahead and use the
> > denaturation conditions you would use in other techniques.  One important
> > note however, at least one of the FPLC size exclusion columns I used many
> > years ago had an affinity for hydrophobic groups.  I never got any protein
> > back when I put it on under denaturing conditions but it would come off
> > when
> > I flushed the column with water.
> >
> > Michael
> >
> >
> >
> > In article <7lcD2.537$xv.5365831 at WReNphoon2>,
> >   ross_turbyfill at wrsmtp-ccmail.army.mil wrote:
> > > Does anyone have a protcol for performing FPLC separations (size
> > exclusion)
> > > under denaturing conditions, i.e., in the presence of guanidine HCl,
> > urea,
> > > DTT, etc?  Specifically, are the concentrations of the buffers used in
> > the
> > > chromatography the same concentrations used to denature the proteins
> > (i.e.,
> > > 8M Urea or 6M GuHCl)?
> > >
> > > Any advice anyone has to offer would be greatly appreciated!!
> > >
> > > ross_turbyfill at NOSPAMwrsmtp-ccmail.army.mil
> > >
> > >    -**** Posted from remarQ, Discussions Start Here(tm) ****-
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> > >
> >
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