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Expression of SeMet labeled proteins: Difference between revisions
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Several methods are available for the bacterial expression of semet-labelled proteins. | |||
In the literature: | |||
Original literature source for met synthesis silencing (for use with semet labelling of non-auxotrophs): | |||
Van Duyne et al. J. Mol. Biol. 229, 105–124 (1993). | |||
Protocols for both auxotrophs and non-auxotrophs, as well as some other non-bacterial systems: | |||
Doublié, S. (1997). Methods Enzymol. 276, 523-532 | |||
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 | |||
Online: | |||
Two protocols for semet labelling as of 11/03/2008: | |||
http://www.doe-mbi.ucla.edu/local/protocols | |||
Another non-auxotroph protocol is copied below, courtesy of G. Fritz. 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: | |||
___________________________________________________________ | |||
Protocol: | |||
Inoculate 2 l of the following media with 8 ml of seed | Inoculate 2 l of the following media with 8 ml of seed | ||
culture (grown for 4 hours at 37°C in 2xYT): | culture (grown for 4 hours at 37°C in 2xYT): | ||