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BN-PAGE of purified membrane associated protein

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BN-PAGE of purified membrane associated protein

Postby Papaver » Wed Nov 16, 2011 12:28 pm

Hello everyone,

Since native page never worked with my protein I started to run BN-gels as it is supposed to be a good tool for hydrophobic proteins. I'm using gradient gels (3-16 %) because the protein seems to form high aggregated complexes.
There is still one problem. I got protein smears so that I cannont perform Western blots. Attached you find an example. In the first two lines I loaded two different markers; first is BSA, second JackBean Urease. Then different protein samples follow. As you can see only BSA makers shows a nice distinct band.

I found several protocols and tried so far the one from Wittig et al (http://www.nature.co...ot.2006.62.html) and lately the one attached.

Since I have no real experience with native pages I would appreciate any suggestion to get rid of the smears.
Attachments
protocol.JPG
111104_BN-PAGE.jpg
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Postby JackBean » Wed Nov 16, 2011 4:59 pm

ah, you have used my urease :o)
I have never done blue-native-PAGE (but I probably will have to), but when I've done native PAGE, I've seen some smear as well, but only in some samples. So maybe you have problem only with the samples, not with the protocol.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby Papaver » Wed Nov 16, 2011 8:00 pm

That's funny :)

But can I do anything to improve the quality of my samples. Someway I hope it's the protocol and not the sample because then I cannot change that much. I was thinking to change the buffer (currently Epps buffer) but then I'm afraid my protein is going to precipitate.
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Postby daniel.kurz » Wed Nov 16, 2011 8:55 pm

I noticed that your sample doesn't have any form of buffer in it which may be charging your protein to aggregate it. I'd look into that.
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Postby Papaver » Thu Nov 17, 2011 7:40 am

I guess you are talking about the sample buffer? The first time I used buffer containing coomassie brilliant blue (CBB) G250. Later I read that it is not necessary for purified proteins. The CBB of the cathode buffer is supposed to be enough to charge the protein. And in fact I do not see any differences between the the loading buffer containing CBB or containing PonceauS. That's why I think there must be something else. Another thought was the lack of a stacking gel.
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Postby daniel.kurz » Thu Nov 17, 2011 3:12 pm

At the percentage of acrylamide that is in your gel, I suspect that it isn't your lack of stacking gel. That would resolve in the early segment of your gel. Do you know the PI of your protein so that you can estimate the charge state of it?
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Postby Papaver » Thu Nov 17, 2011 4:07 pm

The pI is around 5.2.

Tomorrow I will run several native gels with different samples of the purified protein. I also did runs with the same sample but different amounts loaded onto the gel but as expected that wasn't helpful. In general I load around 10 - 20 µg.
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Postby daniel.kurz » Fri Nov 18, 2011 4:07 pm

Your PI is at 5.2 and your buffers are for 7-8.5 pH? Your protein is probably aggregating because its being run in a buffer that is making it negatively charged. It may be interacting with the cross linkages of the gel or even binding to itself.
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