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
