You can also try putting a different affinity tag on the other
terminus, and use that as a second step.
JPK
On Wed, Feb 15, 2012 at 11:25 AM, Xiaodi Yu <[log in to unmask]> wrote:
> Hi Sivasankar:
>
> Are you sure it is due to the protein degradation? Maybe you can try to do a
> western blot or others to check if it is the product of degradation. By the
> way, where you put the 6 histag, N- or C-terminal? If it is at the N
> terminal, maybe it is the truncation version of your protein.
> After looking at the gel, it seems your sample was over-load or had lots of
> unspecific binding to the column. Maybe you can add salt (250 mM NaCl, final
> concentration) and small amount of imidazle in the sample before you load
> onto the column (for example, 20 mM Imidazole final concentration).
> One small "trick" you can try is wash the cell with the buffer containing
> PMSF once before lysising the cell.
>
> Yu Xiaodi
>
> ________________________________
> Date: Wed, 15 Feb 2012 18:39:19 +0530
> From: [log in to unmask]
> Subject: [ccp4bb] protein degradation
> To: [log in to unmask]
>
>
> Dear All,
>
> Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi
> domain) DNA binding protein, that we are expressing at 18 degree Centigrade
> in E. Coli. The protein appears to degrade during purification; we have
> protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM
> EDTA and 1mM DTT throughout during purification ( right from lysis
> stage). We handle the protein at 4 degree Centigrade.
>
> Can you please suggest what precautions we can try to avoid such degradation
> ?
>
> Please find the attached gel picture regarding protein
>
> Sivasankar Putta
>
>
>
--
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: [log in to unmask]
*******************************************
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