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 <CCP4BB@JISCMAIL.AC.UK> (on behalf of Vicky Tsirkoni <vicky.tsirk...@pharm.kuleuven.be>) >Subject: [ccp4bb] >To: CCP4BB@JISCMAIL.AC.UK > > 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: vicky.tsirk...@pharm.kuleuven.be > > ---------------------------------------------------- > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on > behalf of Prerana G. [tracy...@gmail.com] > Sent: Monday, February 24, 2014 5:23 PM > To: CCP4BB@JISCMAIL.AC.UK > 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
