Hi,
I'm sure this question has been addressed before and I
apologize for asking it again. I'm working on a protein
predicted to have multiple disulfide bonds. The protein is
expressed in an E. coli strain with an "oxidizing" cytoplasm,
and is purified under nonreducing conditions (no DTT, b-
ME...). I'd like to produce some Se-Met incorporated protein
for phasing. Given that Se-Met can oxidize, its anomalous
scattering is dependent on its oxidation state, and a mixture
of oxidized and reduced Se-Met makes phasing difficult, what
is the suggested protocol to follow in my situation? Here's
hoping that the solution is not dumping a bunch of Se-Met into
all the purification buffers...
Best wishes,
Reza
Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY 10031
Tel. (212) 650-6070
---- Original message ----
>Date: Thu, 27 Feb 2014 11:22:10 +0000
>From: CCP4 bulletin board <[log in to unmask]> (on behalf
of Vicky Tsirkoni <[log in to unmask]>)
>Subject: [ccp4bb]
>To: [log in to unmask]
>
> Hi Prerana,
> You could try 4°C as well (dont forget to use
> lower protein concentration) in my case this worked
> better.
> Cheers,
> Vicky G. Tsirkone, MSc
> Laboratory for Biocrystallography
> Department of Pharmaceutical and Pharmacological
> Sciences
> KU Leuven
> O&N II Herestraat 49 - box 822
> 3000 Leuven | Belgium
> Tel.: +32 16 3 23419
> e-mail: [log in to unmask]
>
> ----------------------------------------------------
>
> From: CCP4 bulletin board [[log in to unmask]] on
> behalf of Prerana G. [[log in to unmask]]
> Sent: Monday, February 24, 2014 5:23 PM
> To: [log in to unmask]
> Subject: [ccp4bb]
> Hi Vicky,
> I tried to change the ratio of paraffin:silicon oil
> to 1:2, but still the protein precipitated. So I
> tried to do it other way round, I kept
> paraffin:silicon oil ratio as 2:1 and so far it has
> not precipitated. Apart from that, I separately used
> the two oils.In silicon oil there was immediate
> precipitation, but none in parafin oil.
> Prerana
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