Problems with gel filtration

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Tue Apr 23 21:36:41 EST 2002


Hi,

In addition to the useful replies posted by other folks, here's some more :)

You did not tell us what size is your protein, what size was the column and
what total amount of protein you loaded. Therefore, it is hard to say what
*really* went wrong but we can make some estimates with the knowledge of
commonly made errors:

1) you overloaded the column.
2) you ran the column too fast. If you used Sephacryl, unless you run a
26/60 at ROOM TEMPERATURE 1 ml/min is TOO FAST. If it was Sephadex, you may
have been OK depending on the temperature, viscosity of the sample, column
size, etc.
3) you had severe aggregation and cross-polymerization
4) you crushed the bed (see note 2, if you ran your column too fast you may
have overpressured it - if that's the case then you need a new column).
5) you used wrong type of sizing resin (see my note on not knowing what the
protein m.w. was and what the column type was)

If you post the unknown variables, we might be able to help better

A.G.E.






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