Protein Purification : HPLC or FPLC

Wed Mar 30 14:18:53 EST 1994

Hi netters,
in <1994Mar30.170606.5024 at>
Susan wrote:
>Some crystallographers seem to think that the HPLC columns
>are rougher on the proteins, that there is a possibility of
>losing the protein, that the columns clog eventually and have
>to be replaced at high cost.
>Some biochemists (and the HPLC salespeople, of course!) say that
>HPLC can do everything FPLC can, with higher resolution and in
>shorter time.
* Well, that all depends on your protein. *
Of course this might be the answer you have heard lots of times before.
We didn't have problems with the proteins after a HPLC run. They are
cleaner because an HPLC usually can have a better resolution than an FPLC.
In my case (last steps in an Fab purification) I chose the FPLC
because the amounts that I can load on a column are vastly larger
than on our HPLC columns. The separation (size exclusion) is not perfect
but good enough for nice crystals.

For RNA purification we are using the HPLC but with custom made
columns that are quite expensive.
I hope that helps a bit.


        Agustin de la Calle
        Dana-Farber Cancer Institute
        Harvard Medical School
        Boston, MA 02115

        agustin at

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