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Blue native PAGE problems - (Jan/07/2011 )

I am trying a blue native gel for the first time. I'm basically following the Wittig et al. procedures; a copublication with hermann schagger who played a large role in the development of native electrophoresis as far as i can tell. I use a 3-8% tris-acetate gradient gel; it has no sds and a neutral ph. I also used tris-glycine running buffer. These are from bio-rad and are bio-rads recommendations for native gels. My running buffers also contain imidazole and coomassie as per the instructions from wittig et al. They maintain that buffers should be at ph ~7 if made with ph-adjusted imidazole. This is not the case; it's more like ph 8, and i have to use concentrated hcl to acheive neutral ph. I run the gel for 2h at 200V at 4C; the voltage, time, and temperature are appropriate according to wittig and bio-rad. After run, wittig et al also maintain that i should be able to visualize bands. I do not, so i performed a coomassie stain for one hour. After that, i can see bands at the bottom and faint smears in the lanes. Ive included the image. The middle lanes are my samples; the other four lanes flanking them are all protein standards, all from bio-rad, the outer two are the standards i normally use for sds-page, the inner two are the two ladders recommended by bio-rad for this gel. As you can see, bands seem to be moving through gel without separating or are denaturing. Im not sure which. Any well thought out ideas would be appreciated.
Attached Image


If I remember correctly, Bio-rad does not sell native ladder. Try to use a native ladder. Check with Invitrogen and sigma-aldrich.
I had done a few runs of blue native page, however I also unable to visualize my band on gel. I did observed my bands after silver stain. I'm using Bis-tris buffer and not imidazole buffer.
Also, I remember I had to run till ~3 hours at 200V in a mini gel on ice.

In my opinion, I do think that tris-acetate and glycine buffer is not that suitable for BN-PAGE. If you check back with the Wittig procedure, the buffers were imidazole and ions were tricine. Lets see if you can get a better resolution by using tricine rather than glycine.

-adrian kohsf-