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While staying on the subject, I found the following paper very useful when trying to produce Se-Met labeled protein in HEK293 Flp-In. Barton et al. Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells. Protein Sci (2006) vol. 15 (8) pp. 2008-13 Vangelis -- Vangelis Christodoulou Analist C Anastassis (Tassos) Perrakis lab The Netherlands Cancer Institute (NKI) B8.038 Plesmanlaan 121, 1066 CX, Amsterdam The Netherlands tel: +31 (0)20 512 1963 fax: +31 (0)20 512 1954 Website: xtal.nki.nl/Tassos_group/ On 4 Nov 2008, at 03:27, Artem Evdokimov wrote: > http://www.xtals.org/pdfs/selenium.pdf > > (sorry, for some reason first email went out w/o the hyperlink) > > Artem > > -----Original Message----- > From: Artem Evdokimov [mailto:[log in to unmask]] > Sent: Monday, November 03, 2008 9:26 PM > To: 'Jacob Keller'; [log in to unmask] > Subject: RE: [ccp4bb] Semet in non-auxotrophic strains (summary) > > By popular request I've placed our modified Se-Met method online: > > > -----Original Message----- > From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf > Of Jacob > Keller > Sent: Monday, November 03, 2008 8:13 PM > To: [log in to unmask] > Subject: Re: [ccp4bb] Semet in non-auxotrophic strains (summary) > > Hello all, > > Prompted by Kay Diederichs, herein follows a summary of the responses > gleaned from the CCP4BB. The wiki has also been edited to include the > summary: > > http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_Se > Met_labeled_proteins > > Summary: > > 1. Protocols for both auxotrophs and non-auxotrophs: > Doublié, S. (1997). Methods Enzymol. 276, 523-532 > > 2. Two protocols for semet labelling: > http://www.doe-mbi.ucla.edu/local/protocols > > 3. Another non-auxotroph protocol. G. Fritz says this recipe could > probably > be made in one vessel for immediate use, except for the aromatic > aa's which > might dissolve better at basic pH: > http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_Se > Met_labeled_proteins > > 4. Original literature source for met synthesis silencing (for use > with > non-auxotrophs): > Van Duyne et al. J. Mol. Biol. 229, 105-124 (1993). > > 5. Autoinduction semet protocol (allegedly complicated recipe): > Studier FW (2005) Protein production by auto-induction in high density > shaking cultures. Protein Expr. Purif. 41, 207-234 > > Jacob Keller > > > ******************************************* > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > Dallos Laboratory > F. Searle 1-240 > 2240 Campus Drive > Evanston IL 60208 > lab: 847.491.2438 > cel: 773.608.9185 > email: [log in to unmask] > *******************************************