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
