Thanks everyone for your response.
The most likely answer to my problem is protein overloaded onto the
column. I pushed my protein concentration further down to 0.5ml
instead of the usual method, which to run multiple times on SEC.
Adding NaCl in the buffer may also help, as it seems that the
broadness of the elution was affected by addition of NaCl.
I don't think the column is knackered because the previous (and
succeeding) runs of other protein samples were okay.
Different -mer state is a possibility, but something that I am not
really leaning towards to, since, I managed to purify the protein
before in a good elution size.
Science mystery solved.
Thanks again!
Allan
Quoting Filip Van Petegem <filip.vanpete...@gmail.com>:
worst-case scenario for crystallization purposes:
9. Your protein runs as a mixture of monomers, dimers, trimers,
whatever-mers...
Filip
On Sun, Aug 28, 2011 at 7:24 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
>
--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3
phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/
--
Allan Pang
PhD Student
G35 Joseph Priestley Building
Queen Mary University of London
London
E1 4NS
Phone number: 02078828480
