Derek - In the past I've had a similar problem, high amounts of lipid were
associated with the recombinant protein following cell lysis. What has
worked well in the past was to precipitate the protein with ammonium sulfate
then resuspend in the chromatography buffer. Failure to remove the lipid
usually resulted in very poor chromatography.
Scott Leigh, PhD
Cohesion Technologies
2500 Faber Place
Palo Alto, CA 94303
Phone: (650) 320-5581
Fax: (650) 320-5511
email: sleigh@cson.com
> -----Original Message-----
> From: Derek.Ellison@Medeva.com [SMTP:Derek.Ellison@Medeva.com]
> Sent: Monday, February 07, 2000 6:44 AM
> To: Recipients of ABRF List
> Subject: lipid/protein analysis
>
> 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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