electrophoretic mobility shift assay (EMSA)

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Death Adder
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electrophoretic mobility shift assay (EMSA)

Post by kk » Fri Nov 08, 2013 12:21 pm

I am doing EMSA and after the first try having success, I could never reproduce my results: following the streptavidin-biotin-HRP detection, there are no bands on the blot paper.

Compared to the first succesful try, the most striking difference I experience is that while at first, there was a bromophenol blue front formed during native PAA gel elfo, and since then (using the same buffers, equipment, settings) I don't see the blue front line at all! Also, the RioRad gel running tank shuts down every few minutes saying "arcing detected". Then I stop the current and restart, it runs for a few mintues and shuts down again and so on. Randomly, this problem disappears but still, my samples won't sink into the gel fast enough and by the time they migrate into the gel the blue front is nowhere...

Do anyone have a good tip what went wrong?

(I use 6% non-danaturing PAA gel without a stacking gel, I pre-run it at 100V for 1h, I rinse the wells before loading, I don't vortex the samples, the loading buffer doesn't contain SDS either)

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Inland Taipan
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Post by JackBean » Mon Nov 11, 2013 1:50 pm

This looks like problem with elfo. Do you have only one cell? Could you try it somewhere else?
What do you mean by that you cannot see the blue front? It cannot just disappear. Did you try to stain your gel with silver/Coomassie?

Cis or trans? That's what matters.

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