Nian,
Before you dump the column, clean it and run some protein standards on it. If everything looks OK, run a small sample of your protein again. If it behaves the same way, then you may have a protein with hydrophobic patches. Anomalous binding to and elution from polymeric SEC media (sepharose, superdex, etc.) occurs with hydrophobic proteins, including some membrane proteins. This is how they discovered hydrophobic chromatography. If you equilibrate your column in low salt, but your protein is in high salt (as might happen right after a Ni-chelation column), some of it will bind to the column. As the salt concentration decreases, it will release off the column, spreading the protein peak over a wider volume. However, it may still weakly bind to the column matrix and spread out even more.
If this is the case, some remedies would be to vary the salt concentration (lower the better), add in detergents, or use a chaotropic salt like LiCl instead of NaCl.
Good luck,
Michael
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R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
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On Aug 28, 2011, at 5:35 PM, Nian Huang wrote:
Hi, David,
What is the common cause of knackered SEC column? Will equilibrizing a buffer containing 150 mM NaCl directly into a 20% EtOH or vise versa cause the problem. There was no problem just after packing the column.
Nian
On Sun, Aug 28, 2011 at 9:24 AM, David Briggs
<[log in to unmask]> wrote:
Following on from Roger's fine suggestions:
8. Your column is knackered. Can you see fine lines or cracks in the
column? Good packing is v.important for SEC columns.
HTH,
Dave
============================
David C. Briggs PhD
Father, Structural Biologist and Sceptic
============================
University of Manchester E-mail:
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On 28 August 2011 10:25, Allan Pang <
[log in to unmask]> wrote:
> Hi there everyone,
>
> What does it mean when you have proteins eluting in almost the whole column
> volume of S200?
>
> I ran a gel with fractions from 8ml to 20ml and saw band for my protein all
> throughout.
>
> Judging peaks on chromatogram is not useful as it doesn't have any aromatic
> rings.
>
> Cheers,
>
> Allan
>
> --
> Allan Pang
>
> PhD Student
>
> G35 Joseph Priestley Building
> Queen Mary University of London
> London
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>
> Phone number: 02078828480
>