bacterial expression/protein purification

[Ruth McMahon]

Hello,
I am working with the pBAD bacterial his tag expression system from
Invitrogen, and am trying to purify the his-tag protein. I have checked
a number of methods to lyse the cells to release the protein, but in
each case the vast majority of my protein stays in the pellet. I have
used lysosyme, freeze thaw, sonication and NP-40 (NP-40 followed by
freeze thaw seemed to work the best). I even tried 8M urea, even though
I'd prefer to use native conditions. Does any one have any
recommendations of how to generate a lysate which will contain the bulk
of my protein?
Thanks for any help,


Ruth.

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Ruth McMahon, Ph.D.,                                Tel: 353-1-706-2815
Biotechnology Centre,                               Fax: 353-1-269-2016
University College Dublin,
Belfield,
Dublin 4,
Ireland.


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