Protease activity

Michael Witty mw132 at mole.bio.cam.ac.uk
Sat May 5 08:36:04 EST 2001


Dear Richard,
            are you purifying the protein from E. coli?  Which protease
copurifies in IMAC?  Mike.


On Sat, 5 May 2001, Richard Buick wrote:

> Hi all. can anyone help?
> I'm expressing recombinant soluble proteins (His-tag) and purifying them by
> IMAC. The problem is the proteins are being degraded very fast (within 24
> hours) by proteases. I include PMSF and aprotinin in the lysis solution but
> these do not inhibit metalloproteases. To inhibit these I need to add EDTA
> which will strip the IMAC column! The problem is intensified because I
> suspect the metalloproteases are being co-purified on the IMAC column with
> the hi-tagged proteins.
>
> Any helpful suggestions to overcome this problem would be gratefully
> received.
> Please e-mail me as I can only check this occasionally.
>
> Rick
>
> --
>
>
>
> ***************************************************
> Richard J. Buick
>
> E-mail: mail at richardbuick.com
> URL: http://www.richardbuick.com
> Tel(GSM): +44 7801 416792
>
> "Light creates the shadows"
> ***************************************************
>
>
>
>
>




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