RNA/Protein UV crosslinking (UVXL) assay
Peter Ellis <
pjie2@...>
2013-04-26 09:23:35 GMT
Dear all,
I am interested in an mRNA that has been described as being bound to the
cytoskeleton. There are two published papers showing that a labelled
fragment of the 3' UTR can be crosslinked to various (unidentified)
proteins. I am hoping to identify the proteins and characterise them
further.
Unfortunately I am having great difficulty in replicating the original
findings. In our hands the UVXL experiment only gives very low intensity
signal, the band sizes are different to the published work (though I note
that the two published papers also reported a range of band sizes!), and
in our hands the binding cannot be competed by antisense oligos, so it's
not a sequence-specific interaction).
The UVXL binding assay uses a protein extract made with a cytoplasmic
extraction buffer (CEB). Then, a labelled RNA probe (e.g. DIG or biotin
labelled) is added, allowed to interact with the proteins in the presence
of competitors (e.g. heparin, tRNA, other irrelevant unlabelled RNA). This
binding step also uses CEB as the buffer. After binding, the RNA is
UV-crosslinked to the protein, followed by RNase treatment to remove
unbound probe. Finally, Laemmli buffer is added and the whole lot goes on
a Western blot.
CEB composition: 10 mM Hepes, pH 7.5, 3 mM MgCl2, 14 mM KCl, 5% glycerol,
0.2% Nonidet P-40, 1 mM dithiothreitol with freshly added protease inhibitors.
Question 1: Surely this buffer will only extract cytosolic proteins, and
not the cytoskeleton? If the mRNA really is cytoskeletally-bound, then
potentially the binding seen in the original papers represents binding to
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