Author Topic: Second dimension problem  (Read 2431 times)

Violeta G

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Second dimension problem
« on: November 29, 2013, 09:43:29 AM »
Hello to everybody!

I'm come to you looking for some help, since in the last three experiments I've been having a problem with my 2D-SDS-PAGE gels. My 2nd dimension electrophoresis uses to last about 8 hours (gels: 12.5%, 1.5mm; electrophoresis conditions: 15mA/gel about 40minutes + 30-35mA/gel about 7 hours, until the dye front reaches the end). However, in the last weeks my electrophoresis was quite faster: only 6 and 4 hours. After staining the gels, I could observe that the proteomic profile (which I know quite well) was much concentrated and higher than usual, what makes me think it was the dye front who was running much faster, but not the proteins. Giving that, I did one more 2nd dimension, using the same current conditions, in which the dye front reached the bottom of the gel in just 4 hours, but I kept the electrophoresis 3,5hours more. The image of the gel shows that the protein profile had run til its correct position (even further!) but with a quite ugly aspect.

I've read about it in the forum, but I haven't been able to find out any solution. Last time I performed the electrophoresis at room temperature, with a typical Laemmli buffer (Tris-Glicine-SDS; no HCl) and fresh solutions and buffers.

I've attached an image of a normal experiment (slide 1),of the 6hours run (slide 2), of the first 4 hours run (slide 3) and of the last electrophoresis I've referred (slide 4)

Has anybody an idea of what's happening? I do appreciate any help!


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Re: Second dimension problem
« Reply #1 on: November 29, 2013, 12:08:27 PM »
Hello Violeta,

just looking at your gel images, it reminded me of what I once saw caused by upper buffer chamber leakage. Do you run your gels on a rig that has separate upper and lower buffer chambers, like the DALTsix? We've had trouble at one point that the gasket that should make a seal around the glass plates was starting to leak. The gels looked a bit like what you are seeing, with the spots being drawn out to the bottom.
Could also be (combined with) SDS depletion, which you can avoid by adding more SDS to your (upper) buffer, or just preparing your running buffer at 2x concentration.

Hope that helps.

Violeta G

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Re: Second dimension problem
« Reply #2 on: December 10, 2013, 12:58:35 PM »
Hello Elke!

Thank you very much for your reply.

I've tried what you proposed. I've also tried by preparing new solutions of every product as well as using the reactives of another lab; I've also tried by using a new strip on the 1st dimension. But the problem remains: my well-known 2D profile appears as if the gel were too much concrentrated than usual. What strikes me the most it's that the MW markers do not suffer any disturbance in their migration: they appear always in the same position of the gels.

I was thinking about some kind of problem concerning the equilibration step that makes more difficult the entrance to the SDS-acrylamide gel (by the way, I equilibrate in 2%DTT for 15min and in 2.5%IAA for another 15min; my equilibration buffer is 6M urea, 30% glycerol, 2% SDS, 50mM Tris–HCl, pH 8.8).

Any advice? Thank you!