[ccp4bb] agarose-acrylamide composite gels
Hello everyone: 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... Thanks a lot! Cheers, Alex
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
Re: [ccp4bb] agarose-acrylamide composite gels
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:[EMAIL PROTECTED] On Behalf Of Dima Klenchin Sent: Sunday, August 24, 2008 11:34 AM To: CCP4BB@JISCMAIL.AC.UK 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
Re: [ccp4bb] agarose-acrylamide composite gels
Hi Alex, Such gels were used to separate in the past 70S ribosome, plus/minus one protein - so separation is excellent. Cannot recall the references but papers published in the 70s' I believe. The agaose is used to provide "strength" to low percentage acrylamide, say 3% acrylamide/0.5% agarose. Just need to cool the agarose before adding it to the acrylamide. Daniel > Hello everyone: > > 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... > > Thanks a lot! > > Cheers, > Alex >
