NATIVE PAGE

[Harold E Smith]

Dear Lisa-

The simplest solution may be to try it without a stacking gel.  I haven't
done it with native gels before, but it often works for denaturing gels
that exhibit the same problem.  An alternative approach would be to run a
denaturing gel and attempt to renature your protein fragments prior to
assay, but that may not be possible.  Good luck.

-Harold Smith

> Dear all,
> 
> I've been trying to run a 280 KDa protein on a native polyacrylamide gel and 
> have had problems getting it to actually enter the separating gel. The 
> protein comprises of two 140 KDa subunits that I have digested with trypsin 
> to yield 140, 94 and 45 KDa fragments which I have then subjected to native 
> page. I can't use SDS as I am looking at the activity of these fragments in a 
> gel overlay experiment. I have tried altering the % of the gel but to no 
> avail; I still get squished bands at the very top of the separating gel that 
> has barely left the stacking. I've heard that agarose can be used in a native 
> gel to overcome problems with large proteins not entering (although I didn't 
> think my protein was that big to cause such problems!)and if so can anybody 
> send me a protocol.I would be extremely grateful for any other suggestions.
> 
> Thanks,
> 
> Lisa (bmbldo at leeds.ac.uk)  
> 
>