Problems with gel filtration

Frank Fürst ffrank at rz.uni-potsdam.de
Tue Apr 23 11:18:19 EST 2002


Erin <enwarren at unity.ncsu.edu> schrieb:

> So I have been having problems running my gel filtration column.  It is a
> High Resolution column put out by Amersham Pharmacia.  Before loading my
> protein I equilibrate the column with 3 column volumes of buffer.  This
> buffer among other things contains 150mM salt per the instructions to
> eliminate nonspecific interactions of the protein with the column.  Then I
> load my sample which has been concentrated down to under 1 mL and run the
> column at 1 ml/min.  I collect about 2 mL fractions.  When I run an SDS-PAGE
> gel of these fractions I see proteins of all molecular weights in each
> fraction, reflecting poor separation.

Did you also record the UV absorption of the eluate? Where there
separated peaks or a big mess? Where are the peaks, if any?

> It's as if I never put the protein
> sample through the column.  I feel like it must be something I am doing
> wrong and not the column because at least one other member of my lab has
> used this column and achieved nice separation.  Any thoughts on what the
> problem might be?

Two thoughts:

- Perhaps your protein is an oligomer which is in equilibrium with its
  monomers (or lower oligomers). If the equilibrium is very fast
  compared to the separation time, you'll get one peak at an
  intermediate MW. However, if the equilibration time is comparable to
  the separation time, you'll get peak broadening or merging peaks. What
  is the expected MW of your protein, and what column exactly did you
  use? 

- What does "concentrated down to 1 mL" mean in terms of total protein
  concentration? I'm not sure, but SEC is usually used to polish
  proteins that are yet rather pure. It might well be that also a gel
  filtration column can be overloaded, I would have to think about that
  or look it up.

HTH, Frank
-- 
> But I don't really see running SETI at Home as practical as Folding at Home.  What,
> exactly, would be the benefit of finding intelligent aliens on the other side
> of the galaxy?
Maybe they're broadcasting the principles of protein folding... [from bionet.*]




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