Also, for very large proteins you should not boil the samples - if you must
heat, just go to 50-60C for a few minutes. You also may have better luck
with CTAB-PAGE rather than SDS-PAGE (CTAB tends to smear less, for some
reason).
Artem
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Dima
Klenchin
Sent: Sunday, August 24, 2008 11:34 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] agarose-acrylamide composite gels
>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
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