infected B2:GFP-expressing plants, but not in the IPed fraction of
GFP-expressing plants, thus showing that dsRNA was isolated
along with B2:GFP in the GFP pull-down experiment.
1. Cast a non-denaturing agarose gel by boiling 1Â HEPES with
1% agarose powder until agarose has totally melted. Allow the
solution to cool to about 60
C and pour it into a gel-casting
apparatus (see Note 7).
2. Once the agarose gel has solidified (allow at least 15 min at
room temperature) place it into an electrophoresis tank filled
with 1Â HEPES at 4
C.
Fig. 2 North-western blot analysis for the detection of dsRNA in total RNA (two
lanes on the left) and IPed (two lanes on the right) fractions. The experiment
shown was performed with GFP (35S:GFP/Col-0)- and B2:GFP (35S:B2:GFP/Col0)-transgenic A. thaliana infected with tobacco rattle virus. (a) Non-denaturating
agarose gel showing equal loading of RNA samples and RNA quality. (b) Northwestern blotting: same samples transferred from gel in a onto nylon membrane
and probed with recombinant B2-Strep. This north-western blot demonstrates
that dsRNA was co-immunoprecipitated with B2:GFP, but not with GFP
Pull-Down of Viral Double-Stranded RNA
315