We are trying to run purified 26S proteasome complexes in Native gel and are having trouble with it. We have tried several different protocols but we end up with one of three results :
1.Protein remains stuck in the well and does not enter the gel
2. Protein Runs out of the gel (I can not find a pre stained native gel marker or standard )
3.Gel brakes apart and we can not run the assay.
Can anyone offer some advice?
Hi Ettel, we did native page electrophoresis before. I tihnk it may help you. I think you have several problems about native gels. 1st you need correct current for your gel. 5uA e.g. You must calculate your current. 2nd we generally use saccarose satining to follow-up. When it arrives to bottom we end the assay. SO we don't need to use gel brakers.
Hey, I've also done it before.
Which % is your gel ? We always used a 4-12 % gel since proteasomes cannot run in high % gels.
we have tried 3.5% and 4% ,I have seen published works speaking of 2-5% gradient but I don't see how this would work without Rinohide since we can barely keep the 3.5% from braking. Ahmet, what are gel brakers?
It is used for your gel to polimerisation and to keep stable. You can use it until your gels become rigid. Then throw it. Because, the gel brakes don't let to go through current into your gel.
We routinely use 4% native PAGE gels to distinguish 20S from both 1- & 2-capped 26S, but we have also used between 3 & 8% as well as gradient gels of 3-8 and 3-10%.
Generally, we run these gels at a constant 90V and +4C. This should start the gel at about 30 mA and end close to 15-20 mA, depending on buffer/gel%/etc. If you want to run at constant amperage, we set ours to 20 mA but then we extend the electrophoresis somewhat.
Under the 90V constant conditions, 180 minutes for a 4% gel should drive the 2-capped 26S into the gel at least about 1/8 - 1/4" from the well. 3% gels run for 150 minutes typically have about 1/4 - 1/2" clearance. I've run these gels at considerably higher voltages & amps, but I prefer the results from 90V.
As far as the gels breaking apart: you're basically working with jelly, so the only advice I can offer is to be gentle! It takes practice and patience, but with 4% gels we almost never have rips/tears/etc and even the 3% gels are strong enough if you move slowly. Once you can handle the gel without tearing it, the greatest concern should be that you don't stretch or deform it while handling, as these can distort your bands or make them appear to have run oddly.
I have run these gels over-night without ever running the 20S off the gel, so I'm curious what conditions you've used to achieve that.
Both BN- and CN-PAGE nicely separate various proteasome assemblies (as I know from own experience); suitable separation gels are 4-13% or 5-13% PA, just as for OXPHOS complexes. You find an excellent reference at http://www.molbiolcell.org/content/17/12/4962.long (Global Organization and Function of Mammalian Cytosolic Proteasome Pools: Implications for PA28 and 19S Regulatory Complexes).
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