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
one, or experimental error (which for the peptide MS range should be well
below 1Da).
2. the range of possibilities is rather large. For starters, since you're
not measuring the full-size m.w. (or if you are, you're not telling us) who
says that your *lower* band is not your 'correct' protein and in fact the
higher '45 kDa' band is not some sort of post-translational modification
resulting in an *apparently larger* m.w. on SDS-PAGE. Furthermore, in some
really odd cases proteins can give double bands on a gel even without
extensive PTM: such as if you have a partially reduced S-S bond in your
protein that runs on a gel in an incompletely denatured form.
3. the presence of two bands (even if one is a contaminant) is by no means a
deterrent to successful crystallization. Just a few weeks ago my lab
crystallized a protein sample that has two major bands in it, about 12 kDa
apart - that was an unavoidable outcome of preparative proteolysis,
performed on the protein in order to get it to crystallize. Diffraction was
fine, structure was solved, and most importantly the protein was found to be
correct -- which, if you have multiple bands, is not always the case. We all
can tell you various funny/scary anecdotes about folks crystallizing the
wrong protein out of a mixture ("but Mom, it looked *pure* on the gel, like
totally!")Good luck, Artem > 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) on SDS PAGE I do find 2 bands (one of 45 kd and other 2-3 kd > less). The same pattern being retained after FPLC. Intensity of both the > bands remain same even after one or two week of storage at 4 degree. Peptide > mass fingerprinting (after trypsin digestion) suggest both are my proteins > except minor difference in some peptide peaks. Is it because of rare > codon/degradation of protein of interest or any other possibilities? Can I > use this mixure for crystallization? > >> ** >> >> With Regards >> >> *****Vikrant >> *** >> ** >> >> >> >> >> >
