protein electrophoresis

Dafydd ddj at mrc-lmb.cam.ac.uk
Thu Apr 27 11:45:48 EST 2000

Yep, SDS-PAGE will almost certainly denature your protein (especially if you
boil as well) and will result in the loss of any non-covalent interactions. If
you want to try and look at this interaction in a gel based system, have you
thought about non-denaturing (or native - whichever is your preference!) PAGE.
It is effectively the same system as SDS-PAGE but your sample, running and gel
buffers do not contain SDS or any other denaturant or reductant (optional!). As
a result, your protein complex, if specific enough and the interaction is
sufficently strong (say Kd uM to mM), should migrate at a different rate than
your individual components, as your complex remains intact. Unlike SDS-PAGE,
which separates proteins by weight, native gels are dependant on charge as well.
I have observed significant changes in mobility upon the removal of the charge
on a single lysine residue.  The only problem with native PAGE is that you may
have to fiddle around with the pH of your gel buffers but I would try the same
pH that you use for SDS-PAGE.

Hope this is of help,


MRC Centre for Protein Engineering,
Hills Road,                                      email: ddj at mrc-lmb.cam.ac.uk

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