Hi,
Am trying to separate OmpF and C of some E.coli on SDS PAGE. using the ORFs of these proteins from WGS estimate protein sizes of approx 40 and 42kDa.
I do precipitate my protein preps before loading, add 8M urea, load on 10% or 12.5% SDS gels and run with low voltage (max 80V) throughout. however I am not able to nicely separate the bands of these proteins. I have tried to enlarge the stacking gel, run with different current settings (from 150V to 60V)etc without getting good results
does anyone have an idea how I could improve separation using this technique (SDS PAGE)?
thanks
if there are only 2kDa of difference it's hard to have a good separation. However you could try to reduce the acrylamide % (8 or 7.5%). Moreover increasing the stacking gel dimension is not so useful in this case: try to do the opposite (less stacking, more running)
Thanks a lot for your answer Emmanuele
The reason I chose a 12% gel is that I quite often read suggestions of other researchers here in researchgate saying that to separate close molecular weights you would need higher % gels .
there is a paper I use as reference, where they have superb separation of OmpF and C of E.coli with a 12% gel: http://jb.asm.org/content/180/15/3917
from ferenci et al 1998
the bands look great with very good separation of OmpC and F. I don't know how they got these results. unfortunately it was not possible to get the detailed conditions from the authors
what would happen, if I would use a low % (lets say 4%) in the stacking gel and then higher % in the separating?
however I will today try a commercial 12% gel from biorad running at 80V and update you
I would not change the stacking gel composition, it's quite useless for your purpose. With higher acrylamide % you will get better separation, but at lower MWs. You have a 40kDa protein, I would not go higher than 12%. I don't know who's who in your gel, but the separation doesn't look so bad. The image in the paper you suggested was modified (probably contrast enhancement) so you can see sharp bands: if you load less sample you can repeat the same trick.
great thanks
attached the who is who of my gel and below , the gel from frerenci et al. what is astonishing, is that superb separation between OmpC and OmpF in ferenci et al's gel, less so the sharpness of their bands. I have never achieved such a seperation, nor have any of my colleagues in E. coli or other enterobacteriaceae
If band assignment is right, I'd definitely try a smaller protein amount on a 8% acrylamide gel. Alternatively you can try a 12% gel with longer running times
Hi Emmanuele
The biorad precast 12%TGX gels did not work at all (see my other query on coomassie staining) on the other hand the bands were smeared at the size of interest. Now I will go back so self made SDS gels and make a 7.5% gel. could you give me advice on Voltage/run time?
Sample buffer consists of
62.5mM Tris-HCl, ph6.8 containing 10% glycerol, 5% β-Mercaptophenol, 3% SDS (there is NO hat bromophenolblue inside)
I stain with
Comassieblue(0.05%), Methanol 40%, H20(50%) acetic acid(10%)
Destaining:
Methanol abs, acetic acid , H2O in higher conc
Thanks a lot
I've only tried such a low acrylamide concentration once, and I used 120-150 V voltage. When I have an unknown sample I usually stop the separation when BF blue reaches the limit of the gel, and I take note of the elapsed time for future experiments. I don't know how you manage to check migration front without BF blue, but you should do the same. I guess a 40kDa protein will not run on the migration front, so if you want to improve the separation you can let some of your sample flow off the gel. In this way you should achieve a better separation, but, as a drawback, bands could broaden.
I wish you all the best for your experiments
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