Sorry for the off-topic question. I am trying to separate by SDS-PAGE really big proteins (>500 kDa), and lower percentage (3-8%) acrylamide gels do not do the trick. Based on literature searches, acrylamide-agarose composite gels seem the way to go. Is anyone willing to share a protocol? I cannot get hold of any of the old journals where this was described at our rather limited library...
There is no need for a protocol. You just make conventional mix but add low melting agarose stock instead of water and keep everything at 35-37C before adding APS/TEMED. Stacker can be 0% AA/0.5% normal agarose (60C at pouring). Unless you must resolve high and low MW on the same gel, I wouldn't bother with a gradient, just find AA% that works (3% AA with acrylamide:bisacrylamide at 20:1 would be my first guess in your case). Run on the cold room with a tank filled to the top or use circulated water cooling.
Dima Dima
