On Tue, 24 Mar 1998 18:11:30 -0600, mcmahan at oncology.wisc.edu (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.
>
Bornstein and Balian,1977, vol.47, p 132-149, but the Saris et al
procedure allows NH2OH cleavage of proteins in polyacrylamide gels.
>: 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
>NH2OHHCl.
>
I'm using LiOH and i get a precipitate. Using the reaction mixture
(after filtration) give a cleaved/uncleaved ratio of < 1/10 (as judge
by SDS-PAGE and Coomassie blue staining). Is it a "normal" result ?
What about the SDS procedure, and does it work with proteins
immobilized in polyacrylamide gel ?
ledantec at bordeaux.inra.fr
