NATIVE PAGE

L.D. Ofner BMBLDO at leeds.ac.uk
Thu Nov 13 07:14:00 EST 1997


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)  



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