: >Hi,
: >
: >I'm expressing a protein in Sf9 insect cells using a baculovirus vector.
: >Upon purification of the 6x his-tagged protein on a nickel affinity column
: >and after Western analysis, I get multiple bands, with the majority being
: >of smaller size and in greater quantity than the full-length protein. This
: >is the case in both denaturing and nondenaturing purification conditions,
: >and is not ameliorated by the use of "protease inhibitor cocktail"
: >tablets, PMSF, and beta-mercaptoethanol in the lysis buffer and in
: >subsequent wash and elution steps. Assuming specificity of the Ab, and
: >that the multiple bands are in fact due to degradation, what can be done to
: >remedy the problem?
: >
: >Thanks for any suggestions.
: >Victor
Victor,
Is your antibody to the tag or to your expressed protein? Also, is the
tag at the C- or N-term. of the expressed protein?
If you're using an anti-his-tag antibody and an N-terminal tag, then you
may be seeing degradation or premature termination. If you have a
C-terminal tag and are using an anti-tag antibody, you once again may have
degradation or false starts at methionine residues downstream of where you
"think" it should start. In either event, if you are using an anti-tag
antibody, the pattern of the bands seen with silver stain or coomassie in
comparison to that seen with the antibody should give you a clue as to
what is going on. If your antibody is to the expressed protein, then its
nature (monoclonal or polyclonal) in combination with an anti-tag antibody
would also help diagnose your problem.
Given what you say, its not clear that degradation is your problem unless
you have coomassie stainable bands that don't stain with anti-tag
antibodies or monoclonals to your protein. Of course, to test you could
try leaving a sample on the bench overnight and see if the bands get
larger :-)
Good luck,
Steve Dahl
