Digestion of Proteins

Maria Lorenzi (mlorenzi@ascu.unian.it)
Wed, 3 Mar 1999 14:24:47 +0100

Hi,
I'm Maria Lorenzi.
I'm trying to digest my protein with trypsin but I'm not successfull.
I digested it with trypsin, under non denaturating conditions, after
reducing and alkylating my protein but it seems to be very hard to digest
it, using the same conditions of digestion, without any reduction and
alkylation.
I tried the digestion of the toxin, without reduction of it, under
denaturating conditions with urea but it didn't work.=20
My toxin has six cysteines and so probably there is a lot of disulfide
bridges which make the protein very closed and so the trypsin difficult t=
o
work. When the disulfide bridges are broken the digestion is more easy
because the more accessibility of the protein to the trypsin.
Please, can you give to me some advises about this subject? About the
proteolitic enzyme to use and about the more appropriate procedure of dig=
estion?
Thank you very much
Maria

"Donde hay Fe hay Amor, Donde hay Amor hay Paz
Donde hay Paz esta Dios y donde esta Dios
no falta nada"

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*
Dott.ssa Maria Lorenzi
Dottoranda in Biochimica e Biofisica
Istituto di Biochimica
Facolt=E0 di Medicina e Chirurgia
Facolt=E0 di Agraria
Universit=E0 degli Studi di Ancona
Via Ranieri 60131 Ancona, Italia
Tel.:+39 71 220 4677
+39 71 220 4681
Fax: +39 71 280 2117
e-mail: mlorenzi@ascu.unian.it