Problems with gel filtration

Erin enwarren at unity.ncsu.edu
Tue Apr 23 10:08:25 EST 2002


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?

Erin Warren


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