FPLC under Denaturing Conditions

.Krimer Alejandro a.krimer at biosidus.com.ar
Fri Mar 5 08:34:57 EST 1999


Dear Michael:

I was looking for the column that you described with no results.
As far I know all of gel filtration media from Pharmacia (and other
companies) have hydroxil or polar groups rendering the media hydrophilic and
not hydrophobic.
The only exception is a new support which is a polymer compose of
divynilbenzene and polystirene.

Alejandro

> -----Mensaje original-----
> De:	mbarnhart at my-dejanews.com [SMTP:mbarnhart at my-dejanews.com]
> Enviado el:	Thursday, March 04, 1999 1:05 PM
> Para:	methods at net.bio.net
> Asunto:	RE: FPLC under Denaturing Conditions
> 
> 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) ****-
> > > > http://www.remarq.com/ - Host to the the World's Discussions &
> Usenet
> > > >
> > >
> > > -----------== Posted via Deja News, The Discussion Network
> ==----------
> > > http://www.dejanews.com/       Search, Read, Discuss, or Start Your
> Own
> > >
> >
> 
> -----------== Posted via Deja News, The Discussion Network ==----------
> http://www.dejanews.com/       Search, Read, Discuss, or Start Your Own
> 



More information about the Methods mailing list