anomalous running of proteins on SDS-PAGE

John Philo jphilo*nospam* at amgen.com
Wed Jan 7 11:19:32 EST 1998

Robert Woodward wrote:
> Dear All,
> I have a problem with a protein that I am purifying from insect cells
> The protein that I am expressing has a calculated Mw of around 80KDa.
> In very small scale cultures the product migrates to a position of
> around 80KDa when resolved by SDS-PAGE and western blotted.  This is
>  the core peptide size and it shows no signs of post translational modification
> that I can tell. Howeve, upon storage and in some cases before storage
> with larger scale cultures the apparent Mw of the band shifts to around 50KDa.
> This shift is not attributable to proteolysis since I get the same size
> band when I probe westerns with antisera to either end of the polypeptide.
> Has anyone seen anything like this before or has any ideas what is happening?
> The protein does have some disulphide bonds so could I be getting odd
> secondary structure forming  which is not denatured even through SDS-PAGE
> conditions?
> Any ideas would be great.
> Email rw200 at cus.cam.ac.uk
> Robert


Since you say that your protein has disulfides, your conclusion that
this band shift is not due to proteolysis is not necessarily correct. 
Your protein could be getting clipped in an interior region, and the
clipped pieces held together by the disulfide(s), such that the clipped
protein would still be recognized by antibodies directed against both N-
and C-terminal regions.

You definitely need to run REDUCING SDS gels.

John Philo, Protein Chemistry, Amgen

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