help-SDS-PAGE of heavy proteins - (Jul/21/2006 )
I´m having some problems trying to resolve proteins of high molecular weight (might well be about 350-400kD or so) in SDS-PAGE gels for WBlotting. I obtain somewhat distorted and broad bands, even for the prestained molecular wheight markers.
I´ve been trying to optimize the standard protocol we use: 5% discontinous SDS-PAGE gels, Tris-glycine running buffer, 150-200V. I think gels are well polymerized, without alterations in the stacking-resolving interphase or anything like that. I tried a tip from a lab mate, to increase bisacrylamide amounts in order to increase consistency of the gel without changing too much resolution (I went up to almost doubling bis total amount), but with no good result. Could be something about running voltage/temperature?
Could anybody help me? Maybe using some alternative system or so? Any good reference manual/on line resource?
Thanks everybody in advance
for long proteins, i use 40:1 mix and run at 80-100V maximum.
Can you use pre-casted 4-20% gradient gel? that gives me very good resolution acrose wide MW ranges.
a while ago i was trying to detect a ~300kDa protein - i got the best results using a 6% resolving gel, no stacking gel, and running at 100V for 5-6 hours (until the highest marker band, 250kDa, had migrated halfway down to the bottom).
Thanks!! Wow, that was a quick answer!
Fred, so you recomend better to increase acryl:bisacryl ratio instead of lowering it?
Thanks a lot again
some time ago, we were separating myosin heavy chain isoforms on sds gels. we used standard 37.5:1 acrylamide:bis ratio. the gel was a 3.5-5% gradient with a 8-0M urea gradient (reverse gradient to the acrylamide). the urea sharpens the bands.
Well, thanks everybody. I´ll play around with it.
Anyway, could you recommend me your favorite reference books/on line resources/whatever for protein electrophoresis? Sometimes I would like to have a better theoric background on the basis of the different techniques, in order to try to optimize for a particular experiment. We´ve got "Protein and Proteomics" (4thed) in the institute, but despite of the good general explanations, it´s not very extense on specific techniques for electrophoresis.
When I was doing my Phd, we used to separate our high MW proteins, we used 6% separating and run at 100 V, but we always checked that we did over run on the gel almost 3-4 h than required time, that worked for us, you can give a try!!