1)My protein has 14 cysteine residues.
>
That can be a big issue in itself :)
> 2)It is not a metal binding protein.
>
Are you sure, with this many Cys? Positive?
> 3)I have added the protease inhibitor cocktail + PMSF during sonication and
> cleavage. I saw only one band of protein bind on b
ealrier on this forum Artem have posted about PTM
in bacteria.
this answer your question on PTM in bacteria.
QUOTE
"Yes, this does happen.
Spontaneous α-N-6-Phosphogluconoylation of a "His Tag" inEscherichia
coli:The Cause of Extra Mass of 258 or 178 Da in Fusion Proteins
http://www.sciencedire
How about incomplete TEV cleavage ?
Your protein is a dimer/multimer in solution and some of it is cleaved and some
not and they stick together and are separated on the SDS gel ?
Jürgen
-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Dear all
Thanks for yours valuable suggestion.
Just some addition information to make the query clear.
1)My protein has 14 cysteine residues.
2)It is not a metal binding protein.
3)I have added the protease inhibitor cocktail + PMSF during sonication and
cleavage. I saw only one band of protein
1. your information is helpful but not enough to tell if this double-band
pattern is a product of e.g. proteolysis or of abortive translation. You
don't specify what these 'minor differences' are - in the world of MS there
is no such thing as 'minor' since every difference is either a significant
o
I assume that the loss of the peptides that you observe for the smaller protein
is at the other terminus from the tag? If it is at the terminus where the tag
was it would suggest that removal of the tag using proteolysis is the most
likely cause. Though saying that Factor Xa/thrombin is not 100%
What are the minor differences in peptides ?
Any PTM's perhaps ?
Jürgen
-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
L
I am working on 45 kd protein (pI 5.3) for crystallization . It is in pGEX
vector.I do the expression (induction 24 degree 16 hrs) and purification from
Rosetta 2 DE3 cells (has additional rare codon).when I run the affinity
chromatography purified protein (cleaved protein after removal of tag
