hydroxylamine cleavage in GuHCl
bassuk at u.washington.edu
Thu Mar 26 01:15:05 EST 1998
Scott is correct in that Li will confer a greater solubility than Na
to your mixture. Please follow the published procedure exactly.
On Tue, 24 Mar 1998, Scott McMahan wrote:
> In article <roney.graf-ya02408000R2403981354130001 at news.uni-konstanz.de>,
> roney.graf at uni-konstanz.de (Roney Graf) wrote:
> : I am trying to cleave a protein at a unique N-G site using
> :hydroxylamine. Since the protein is poorly soluble (I have inclusion bodies
> :from an E. coli prep), I need to perform the reaction in GuHCl.
> What about using SDS? I used it when I did a NH2OH reaction. Look at
> Biochemistry 33/12092-12099/1994
> : There are
> :some references giving a simple protocol for this, but they seem to be
> :lacking some info.
> : Saris et al (Analytical Biochemistry 132/54-67/1983) say: Dissolve 1.1g
> :NH2OH-HCl (2M), 4.6g GuHCl (6M), 15mg tris (15mM) in 4.5M LiOH to get 8ml
> :at pH9.3.
> There's a better reference for the actual cleavage in Methods in Enzymology
> #47. I'm not sure of the pages, but the table of contents should tell you.
> : The problem is, there is no way I get the NH2OH and the GuHCl dissolved
> :together. Separately, each of them dissolves to a cloudy solution which can
> :be clarified by filtration, but together it's just a mess. I'm using NaOH
> :instead of LiOH, but as far as I get it the LiOH is just used because it's
> :volatile and easier to get rid of. Or is it?
> The LiOH is used to prevent the precipitation of NaCl. Since you're
> starting with 8M hydrochlorides, the necessary NaOH to neutralize them
> brings the total concentration of Na+ and Cl- pass the saturation point,
> and you get a NaCl preciptate. Filtering should give you a workable NH2OH
> solution (the Li+ and Cl- ions are just spectators), as will using LiOH
> (LiCl has a higher solubility) or using SDS as a denaturant instead of
> GuHCl (less NaOH needed to get the pH up to 9.3 and only 2M Cl- from the
> Scott McMahan
> mcmahan at oncology.wisc.edu
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