Native-PAGE, help! - (May/24/2013 )
I am trying to separate my protein by using Native-PAGE. My protein has PI 8.2-8.7, MW is 7.6kDa. Should I try which buffer and running condition first?
Just follow the paper which you are citing right now.
if some problems reveal. make a modest modification, then.
I have followed many paper, but they use RP-HPLC. My lab don't have RP-HPLC system so that I want to analyse the protein by using Native-Page. I just need to determine that whether my recombinant protein has the right structure or not by comparing to the commercial protein. My protein has PI > 7.0 so I think I will use acidic native-PAGE (acetate-KOH-Histidine). Because my protein is only 7.6kDa so I don't know whether this method can separate my protein or not.
you have two viable choices for native page: "normal" ornstein and davis (o-d) page (runs at pH 9.5) or acid pH page.
mobility in o-d gels may be low (won't be useful for size determination) but should still be in the correct direction. also, most of the proteins which may be along with your protein will have a pI<7 so it may still be a better choice than acid page.
either way, you'll want to use a 12 or 15% gel.