Problems with gel filtration

[ChenHA]

Erin wrote:
> 
> 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.  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?
> 

Check the time your protein elutes out - i.e. whether it is coming out
at the expected elution volume (you need to calibrate using protein
standards).   I see that occasionally when the proteins are aggregated
which sometimes can be concentration dependent - the protein forms
multimeric aggregates of varying sizes.


> Erin Warren
> 
> <http://www.biowww.net/forum/read.php?f=6&i=999&t=999>