Re: [ccp4bb] Dnase activity in E. coli expressed his tag proteins

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: Dnase activity in E. coli expressed his tag proteins
From: Filip Van Petegem filip {- dot -} vanpetegem {- at -} GMAIL {- dot -} COM
Date: 2007-08-06

Dear Narayanan,

you could simply test that by a negative control: transform your cells with
an empty vector, and follow the same (exact) purification protocol. No DNase
activity there = probably no DNase contaminant in your original prep.

Cheers

Filip

On 8/6/07, Narayanan Ramasubbu wrote:
>
> Dear All:
> This is off topic but I would rather try here first.
> I am wondering whether Dnase could be a contaminant during the nickel
> affinity purification of a his.tag protein expressed using pET29b. The
> cells were disrupted using sonication only. Very high yield (30 mg/liter
> of cell culture). FPLC purification. SDS-PAGE shows a singe band even
> when overloaded. Silver staining is in progress.
> The protein I am interested in does not have any similarity to Dnase.
> However, when treated with supercoiled dna, the dna is degraded as
> efficiently as with Dnase I from Sigma.
>
> I am looking for ways to show that my preparation does not have any
> Dnase contamination. I would appreciate it very much if someone points
> me in the right direction.
> Thanks a lot
> Subbu
>

--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpetegem@gmail.com

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: J. Struct. Biology EndNote style
From: artem {- at -} XTALS {- dot -} ORG artem {- at -} XTALS {- dot -} ORG
Date: 2007-08-06

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