Hi everyone,
I have a big trouble when doing EMSA. I used the biotin-labeled probe to detect protein-RNA complex. however, the whole well path is so smear that I cannot find a clear single band. I have changed the ration between RNA and protein for several times, but they all did not work. I have confirmed proteins are in good quality since I do not have this problem with other RNA probes. Anyone have suggestions?
Thanks a lot.
Hi Ying, some times EMSA does not work for particular probe/protein even if they interact OK in solution, - it is tricky assay. One possibility is that your complex is not very stable at conditions you are trying to run it. Think about reducing or increasing salt, changing pH etc. One trick people do to increase stability of complexes in EMSA is to run it in a cold room ( or in ice box), and it (gel) should be cooled down in advance, so it is +4. Plus, you could load on running gel so that you sample immediately runs into the gel (gel helps to stabilize complexes often). It is tricky to do, one need to use cap-free gel-box. Ask somebody to help you to set it up with electrical lines for old agarose gel-boxes. :) Be careful not to touch buffers in such setup! Try the cold run first.
one of my colleages used to work with minor concentrations of glutar aldehyde to fix and stabilize the complexes after the incubation. At least with radioactive labelled probes this seem to work quite well
Sven Reister stabilizing EMSA complexes post-incubation by crosslinking with glutaraldehyde is an interesting idea; do you happen to have an EMSA-specific crosslinking protocol or perhaps a reference for it?
I am also facing the same problem, and run gel different conditions like pH change, salt concentration, and gel run in cold room, when pH changed then smeared band form but problem not resolve.
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