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I just tried that protocol, and had essentially all of my protein crashed out. Any tips on optimizing the methylation reaction to reduce precipitation? Perhaps reducing the formaldehyde or dimethylaminoborane, shortening the incubation times, etc.? Thanks, Nat On Wed, Apr 14, 2010 at 6:00 PM, Engin Ozkan <[log in to unmask]> wrote: > I was surprised to get a few messages asking for our protocol for reductive > methylation of proteins for crystallization. We employ almost exactly the > protocol published by Walter et al, Structure, 2006. This is a "Ways and > Means" article that made us realize how easy it was to do this regularly on > failing projects, and it has a section titled "The Protocol" for those > wanting to do it. > > A couple of things to add: We use methanol-free formaldehyde. Also, remember > that your protein has to be in an amine-free buffer; Tris is no good during > the reaction, but it can be used to quench the reaction. > > Engin > > On 4/13/10 9:51 PM, Engin Özkan wrote: >> >> Dear Oliver, >> >> In our lab, reductive methylation using dimethylaminoborane is regularly >> performed, and nearly everything we work on have native disulfides. Among >> five or six reactions I've performed on molecules with disulfides, I have >> not had a case where solubility or stability was affected. In one case, >> however, methylation broke up a protein complex (the interface is lysine >> heavy). I did also hear from lab members a few cases where there was protein >> precipitating during methylation, but that seems to be the exception rather >> than the norm. >> >> Engin >> >> On 4/13/10 7:55 PM, Oliver Clarke wrote: >>> >>> Hi all, >>> >>> I'm currently trying to crystallise a two domain protein which contains >>> several structurally important disulfides. We have a high resolution >>> structure of one domain (~1.4 A resolution), which reveals quite a few >>> solvent-exposed lysines, some of which are involved in crystal-contacts. >>> >>> The two-domain construct also crystallises, but the only crystals >>> obtained after extensive optimisation are stacks of thin-plates that show >>> poor diffraction (multiple lattices, streaky spots) to around 3 A. >>> >>> I would like to attempt modification of the lysine residues by reductive >>> methylation or cyclic pentylation (in the hope of improving morphology >>> and/or diffraction), but I am worried that the reducing conditions required >>> for the reaction (due to the presence of the dimethylaminoborane complex) >>> will disrupt the native disulfides and result in protein aggregation or >>> denaturation. Does anyone have any experience with reductive methylation of >>> disulfided proteins, or know of any references describing the same? >>> >>> Thanks in advance, >>> >>> Oliver Clarke. >>> >>> ______________________________________________________________________ >>> The information in this email is confidential and intended solely for the >>> addressee. >>> You must not disclose, forward, print or use it without the permission of >>> the sender. >>> ______________________________________________________________________ >> >> > > > -- > Engin Özkan > Post-doctoral Scholar > Howard Hughes Medical Institute > Dept of Molecular and Cellular Physiology > 279 Campus Drive, Beckman Center B173 > Stanford School of Medicine > Stanford, CA 94305 > ph: (650)-498-7111 >