Problems with expression in E.coli

[Paul Klosen]

>Paul,
>
>By what criteria are you determining there is no expression?  I have a
>his-tagged construct in pET28, and while I found it difficult to see
>induction just by staining, it was very obvious when I ran a western blot
>using anti 6Xhis antibody.  Also, my protein turn out to be insoluble, and
>once I made soluble/insoluble fractions, it was very obvious protein was
>being expressed.  Have you tried these approaches yet.

I did check by Western blot for HIS-tagged proteins (positive control OK, 
constructs negative). These Western blots were done on E.coli total 
proteins, solubilized either in SDS-Page buffer, or in BUST 
(betamercapto-Urea-SDS-Tris).

Paul
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Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur  12, rue de l'Universite
F-67000 Strasbourg, FRANCE
Tel. 03.90.24.05.01  Fax. 03.90.24.05.28
========================klosen at neurochem.u-strasbg.fr=========================

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