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 <[log in to unmask]> 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: [log in to unmask]
