> obtain a signal just at around 90 KD. Does anybody have seen
> something like this and does anyone have an explanation for it. What
> do you think I can do to check that the band on the western
> corresponds to my protein?
Many people have seen this :)
Dependng on the details of your problem, the possiblities are nearly
1) gene error or mutation resulting in premature termination
2) mrna folding resulting in slippage or premature ribosome release
3) proteolysis (including auto-proteolysis!)
what can be done:
1) check your plasmid(s) sequence
2) n-term. seq. your protein
3) MS. with your size, ESI-MS may be stretching it a little, try
4) if direct MS does not work, or if you discover that you don't have
simple proteolysis but say, a piece of protein missing (but not at any
of the ends) you can digest the protein with (e.g.) Lys-C protease
(Available from e.g. Fluka) and
check the fragments on an MS. Then do a cyanogen bromide digest
(methionines) and also check the MS. Solve the crossword puzzle.
Hope it helps.
|Dr. Artem Evdokimov Protein Engineering |
| NCI-Frederick Tel. (301)846-5401 |
| FAX (301)846-7148 |
|eudokima at mail.ncifcrf.gov |