Hi, Deb -
Once again into the breech, dear comrades...
Silica-based ion-exchange materials can tolerate 100% organic solvent, but
that's obviously not practical if your solutes are proteins. Here's a list
of applications and conditions that various groups have used with PolyCAT A,
our silica-based cation-exchanger. All applications feature the same %
organic in both mobile phases:
1) Hemoglobins, bFGF, and other delicate proteins: 0% organic.
2) General polypeptides (PEGylated, etc.) and recombinant proteins < 40 KDa:
20% organic, either EtOH or ACN. This poses no problems with the
high-salt buffer. Selectivity generally improves about 50% compared with 0%
organic.
3) Human growth hormone, interleukin-2, and snake venom proteins: 40% ACN or
PrOH. This increases selectivity 2-4x! However, with levels of NaCl above
0.3 M, you run the risk of phase separation (ACN and water are miscible, but
ACN and salt water are not; the salt increases the polarity of the water to
the point that the relative difference in polarity of water vs. ACN is too
great for complete mixing). The solution is to use salts that are less
structure-forming than NaCl: ammonium acetate, NaBr, NaClO4 (which is
transparent at 220 nm), etc. Specifics:
a) IL-2: See Marchese et al., J. Chromatogr. 504 (1990) 351. They were
separating sialylation variants of IL-2. With > 40% PrOH, they didn't get
complete separation. With < 30%, they didn't get complete recovery. 30%
gave them complete separation and recovery.
b) Snake venom proteins: Ed Hawrot's group (Brown U.) produced
alpha-bungarotoxin in yeast. Problem: the species chosen, Pichia pastoris,
stuck carbohydrate chains on various residues. The native sequence isn't
glycosylated. A gradient with aq. ammonium acetate (50-300 mM, pH 6)
afforded an envelope with a few maxima protruding. Inclusion of 40% ACN
spread the envelope out by a factor of 3, leading to the complete resolution
of @ 15 variants, including the desired (nonglycosylated) product. This work
is in press in Neuroscience.
4) Histones: 70% ACN. See Lindner et al., J. Chromatogr. A, 782 (1997) 55.
Phosphorylation variants of Histone H1 are poorly resolved with an aq. salt
gradient. Inclusion of 70% ACN inverts the elution order and resolves these
variants by several peak widths. The reason is that the use of 70% ACN
superimposes hydrophilic interactions on the electrostatic effects. Since
phosphate groups are quite polar, their presence leads to a considerable net
increase in the retention of the H1 protein. Now, histones are quite basic,
and it's necessary to use a lot of salt to elute them (up to 0.7 M). NaClO4
is handy for the purpose; quite soluble in ACN and transparent at 220 nm.
Just make sure you use a buffering agent that doesn't cause something to
precipitate. A good salt for the purpose is Na-methylphosphonate.
ACN and PrOH seem to be equally effective in most cases (exception: Zinc
zipper proteins were much better resolved using PrOH). Use ACN if you have a
choice because of the high viscosity of PrOH solutions. MeOH and EtOH aren't
quite as effective and higher concentrations may have to be used.
Anion-exchange: Same rules as cation-exchange.
Hope this answers your question. See you in Seattle!
Andy Alpert
PolyLC Inc.
tel: (410) 992-5400
************************************************************
<< Subj: Re: lipid/protein analysis
Date: 02/08/2000 11:19:29 AM Eastern Standard Time
From: mcmillen@morel.uoregon.edu (Deb McMillen)
To: POLYLC@aol.com
CC: abrf@aecom.yu.edu (Recipients of ABRF List), abrf-request@aecom.yu.edu
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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