Andy, How much acetonitrile or EtOH can you put on ion exchange columns--can you
give some specific examples of columns and percentages--with typical salt
concentrations?
Thanks,
Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR 97403
POLYLC@aol.com wrote:
> Derek:
>
> Why not try ion-exchange chromatography with a considerable amount (40+%)
> organic solvent (ACN, EtOH, or PrOH) in the mobile phases? Whether the
> interaction of the (phospho?)lipid with the protein is through electrostatic
> attraction or hydrophobic interaction, this combination should take care of
> it. IEC is a good capture step too; capacity 4x greater than reversed-phase
> (RPC). You can then perform RPC as a second step, during which you both
> desalt the collected protein and perform a complementary purification. Also,
> selectivity in IEC is often at a maximum around 30-40% organic solvent in the
> mobile phase. Contact me if you want some examples.
>
> Best regards,
>
> Andy Alpert
> PolyLC Inc.
> 9151 Rumsey Road, ste. 180
> Columbia, MD 21045 USA
> tel: (410) 992-5400 FAX: (410) 730-8340
> ************************************************************
> << Subj: lipid/protein analysis
> Date: 02/07/2000 4:49:54 PM Eastern Standard Time
> From: Derek.Ellison@Medeva.com
> Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
> Facilities)
> To: abrf@aecom.yu.edu (Recipients of ABRF List)
>
> I wonder if anyone can help me with this - we don't have much experience of
> working with lipid, but we currently have quite large amounts of lipid
> present in recombinant protein samples we are trying to develop rpHPLC
> separations for. How will the lipid affect reversed phase? should we
> delipidate first? (there seems to be a problem with our protein
> precipitating when we try chloroform/methanol washing). Will phospholipid
> (we assume that's what it is) co-elute with protein in gradients of
> acetonitirile on C8/C18 columns? Can anyone recommend any references?
>
> Thanks in advance for any advice,
>
> yours in ignorance of all things lipid,
>
> Derek Ellison
> Medeva Development
> Speke
> Liverpool
> L24 9GR
>
> Phone: 44 151 705 5442
> Fax 44 151 705 5189
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