lachlan harris wrote:
>> Can anyone help me with this problem? I have a pure microbial protein
> which when run on an SDS non-reduced (ie no mercaptoethanol) PAGE gel
> runs at approximately 50 KDa. When the same protein is run under
> SDS-reduced conditions two subunits appear- one running at approx 40 KDa
> and the other at approx 60 KDa.
>> It was my understanding that the two subunits should add up to be the
> same size as the protein run under SDS-non reduced conditons. But this
> isn't happening.
You could spend a fair bit of time sorting this one out. If you have
access to a MALDI mass spec (perhaps in a core facility), the fastest
solution would be to run the protein in the MALDI, with and without
reduction. You'll likely get accurate molecular weights quickly, and
with very little material used.
If you can't get to a MALDI, then there are several possibilities which
come to mind, none all that attractive, but the one I would consider
first is this:
1) You do have a monomer whose true molecular weight is close to 60 kD.
2) Treatment with beta-mercaptoethanol reduces one or more disulfide
bridges, allowing the protein to "open up" and run at 60 kD instead of
50 kD.
3) The 40 kD is an artifact -- a clipped form produced during
beta-mercaptoethanol treatment. There is ample documentation of such
artifacts in the literature.
To reduce the chance of this happening, try adding 1 mM EDTA or DPTA in
addition to the mercaptan (the reactions are generally catalyzed by
trace concentration of transition metals). Also try to minimize or even
eliminate the boiling step.
Go for the MALDI if at all possible.
Rod Levine
NIH
rlevine at nih.gov
