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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