To be specific, elution of the column with 40 mM EDTA.2Na for 24 hours at a
low flow rate. This not only passivates "hot"sites in the frits and column,
but also passivates the HPLC system, rendering it resistant to chloride ion
attack for 3-4 weeks.
This probably accounts for the ghosting you saw with ovalbumin. Unlike BSA,
ovalbumin occurs as phosphorylation variants. If the phosphate groups
interact with the frits via chelation, the protein might well manifest
"ghosting" in subsequent runs. Try this passivation with EDTA.2Na and then
see if you still get ghosting with ovalbumin.
Best regards,
Andy Alpert
PolyLC Inc.
tel: (410) 992-5400
*********************************************************************
<< Subj: Re: Column Clean Up with TFE
Date: 06/08/2000 9:28:19 PM Eastern Daylight Time
From: schooley@med.unr.edu (David A. Schooley)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
At 2:00 PM -0700 6/8/00, NovickL@immunex.com wrote:
>Do you think this would work with columns that are end-capped (like the
Jupiter columns from Phenominex)?
The Vydac columns we used it on were end-capped. Thanks to
Matt Sweeney for the discourse on "new surfaces eat"- yes, even
Sephadex gels. That was when I first learned to use BSA, to prepare
a size exclusion column for use. One time I scaled down the amount
of BSA 5 X over what I was told to use and then found that the lower
molecular weight peptide calibration standards practically
disappeared.
Why BSA? It is cheap, relatively pure, and comes off even a
C18 column at a convenient point with 60% acetonitrile (right near
end of gradient). Tried ovalbumin once and it ghosts forever, which
BSA does not. There may be many things out there that work equally
well but ovalbumin is not one of them! I am happy to stick with what
I know works.
Andy Alpert says column frits contribute much of this
stickiness, and advocates a prolonged flush with disodium EDTA to
kill the activity of frits.
David
--
David A. Schooley
Dept. of Biochemistry/330
Univ. of Nevada
Reno, NV 89557
schooley@unr.edu
tel: (775) 784-4136; fax (775) 784-1419
NOTE NEW AREA CODE: Mandatory after 5/15/99
>>
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