hydroxylamine cleavage in GuHCl

Scott McMahan mcmahan at oncology.wisc.edu
Thu Mar 26 17:23:00 EST 1998


In article <351a522b.7725282 at news.hol.fr>, loick.ledantec at hol.fr wrote:

:Bornstein and Balian,1977, vol.47, p 132-149, but the Saris et al
:procedure allows NH2OH cleavage of proteins in polyacrylamide gels.

Yeah, if you don't mind doing in 5 steps what can be done in 2.

:I'm using LiOH and i get a precipitate.

How's your LiOH?  It can form carbonates, IIRC.  With a fresh batch of
LiOH, I never had problems while using GuHCl, though I did abandon that
denaturant fairly quickly.

: 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 ?

Not for what I did.

:What about the SDS procedure, and does it work with proteins
:immobilized in polyacrylamide gel ?

Check out the reference I gave earlier.  I did the digest _in_gello_.  The
Saris et al. method was made much simpler because you don't have the GuHCl
+ SDS precipitation problem.  Basically, just throw the gel in the solution
of 0.1% SDS, 2M NH2OH, pH 9.0 + buffering agent.

-- 
                                         Scott McMahan
                                         mcmahan at oncology.wisc.edu



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