Problems with gel filtration

[Les Erickson]

How did your molecular weight markers run?

>===== Original Message From enwarren at unity.ncsu.edu =====
>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
>
>
><http://www.biowww.net/forum/read.php?f=6&i=999&t=999>

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