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
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: rmgarav...@gmail.com
****************************************************************
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 <drdavidcbri...@gmail.com
> 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:
david.c.bri...@manchester.ac.uk
============================
http://manchester.academia.edu/DavidBriggs (v.sensible)
http://xtaldave.wordpress.com/ (sensible)
http://xtaldave.posterous.com/ (less sensible)
Twitter: @xtaldave
Skype: DocDCB
============================
On 28 August 2011 10:25, Allan Pang <a.p...@qmul.ac.uk> 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
> E1 4NS
>
> Phone number: 02078828480
>