Bands not getting into the gel

[CKUW]

Hi, I've done EMSAs before with success and I'm not sure what the problem is that I'm having right now with them. I made new TBE buffer, making sure that it's the correct pH, and I'm positive that I'm lysing the samples in enough buffer, so I don't understand why now I'm not getting the bands to actually run into the gel. When I develop the film I get the free probe at the bottom of the gel, but the bands haven't even migrated into the gel, they look to be stuck in the wells or right below where the wells were. Why wouldn't they get into the gel? When the gel is running, I can reliably watch the dye front migrate through the gel to the bottom. It doesn't seem to matter, because the samples don't get into the gel. Any help you could give me would be greatly appreciated. Thanks

[junli]

Hi,
Sorry I can not answer your question, I have the same problem here, instead. Could anybody help u out? that will be really appreciated.

[kmunson779]

Have this probe/protein combo been shown to work before, or are you optimizing a new pair? It's possible the complex is just too big for your current gel conditions; you could try reducing the gel percentage and running the gel for longer. The other old standbys could still apply as well: make sure the comb is scrupulously clean, that the wells don't get any chance to dry out before they're immersed in running buffer.

Are you using radioactive or fluorescent probes?

[junli]

Thank you so much for your reply.

I am trying to detect the effect of my gene (ING4, a tumor suppresor ) on the binding activity of NFKB. basically i am optimizing a new pair. but I think the subunit of NFkB is just about 65 or 50 KD, while my probe is only 25bps long, do you think it may be due to the size too big? My probe is fluorescent labeled and I can always see the free probe at the bottom of the gel.

Thank you again!

[junli]

Thank you very much for you reply. I am testing the effect of my interest gene (ING4, a tumor suppresor) on the binding activity of NFkB, so basically I am optimizing a new pair. But I think as the subunits of NFkB is only 65 or 50 KD and my probe is just 25bps long, do you think it may be due to the size too big? I am using fluorescent labeled probe (Pierce). Thank you again.