weird migration of sds-page

P.J. Shaw pjs14 at le.ac.uk
Tue Oct 21 13:34:14 EST 1997


Hi all,

I have made some proteins using the pMALc2 vector from NEB. I have stuck
homeodomains (60aa) c-terminal to the MBP vector protein. Sequencing has
shown the fusions to be in-frame and stop after the last aa of the
homeodomain. The MBP alone is about 43 kDa, so my fusions should be
slightly larger. When run on denturing SDS-PAGE, the MBP alone runs at the
correct size, but all my constructs show two bands, a minor one about
the same size as MBP and the majority much smaller, about <=40 kDa. When
run on native PAGE, the fusions run as one band slightly slower migrating
than MBP. Fusions cleaved with factor Xa shift to the same size as MBP. I
guess in denaturing PAGE, there is some conformation problem affecting the
migration but I thought that was a problem only for very hydrophobic
proteins like membrane proteins. If anyone has seen this before or can
proffer a simpler explanation, I would be very grateful.

Thank you, 

Philip J. Shaw

pjs14 at leicester.ac.uk




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