5. The leaf disks can now be observed by laser confocal microscopy to detect B2:GFP (GFP channel) and RFP-tagged protein
of interest (RFP channel), in noninfected tissues and virusinfected tissues. The high protein expression levels provided
by the strong gene promoters should allow easy observation
(see Note 17). An example of such an experiment is shown in
Fig. 5.
4 Notes
1. If mass spectrometry analysis is planned, it is highly advisable to
perform at least three technical replicates for each sample/
condition.
2. This step can optionally be repeated for further clearing of the
lysate.
3. This can be done by placing the tube on a magnetic stand and
letting the bead cluster on a side (non-colloidal beads) or by
letting the solution flow through a magnetic column (colloidal
beads), depending on the kit used.
4. The washing method depends on the kind of beads used. If
using non-colloidal beads, resuspend the beads in 500 μL cold
lysis buffer by gentle inversion and incubate them on the
rotating wheel for 5 min, after which the tubes are again placed
on the magnetic stand to remove the beads and the buffer is
removed. If using colloidal beads (such as those in the Miltenyi
μMACS kit used here), let 500 μL lysis buffer flow through the
column by gravity. In all cases, the buffer should be removed
from the beads after the last wash.
5. If using non-colloidal beads, add Trizol to the tube containing
the beads. If using colloidal beads, remove the column from
the stand and pass the Trizol through the magnetic column
into a 1.5 mL tube, gently pushing with a Pasteur pipette if
necessary.
6. If colloidal beads are used for pull-down, some may be carried
over into the final purified RNA suspension. In our experience,
these beads did not interfere with the further analysis.
7. All material used for electrophoresis and transfer should be
clean or treated with soap, rinsed, and cleaned with 70% EtOH.
8. Electrophoresis should continue until the blue dye migrated to
the middle of the gel. Optionally, a dsRNA ladder such as the
Phi6 dsRNA ladder [7] can be used to monitor dsRNA sizes
and their separation during electrophoresis.
9. The membrane must not dry.
Pull-Down of Viral Double-Stranded RNA
325
of interest (RFP channel), in noninfected tissues and virusinfected tissues. The high protein expression levels provided
by the strong gene promoters should allow easy observation
(see Note 17). An example of such an experiment is shown in
Fig. 5.
4 Notes
1. If mass spectrometry analysis is planned, it is highly advisable to
perform at least three technical replicates for each sample/
condition.
2. This step can optionally be repeated for further clearing of the
lysate.
3. This can be done by placing the tube on a magnetic stand and
letting the bead cluster on a side (non-colloidal beads) or by
letting the solution flow through a magnetic column (colloidal
beads), depending on the kit used.
4. The washing method depends on the kind of beads used. If
using non-colloidal beads, resuspend the beads in 500 μL cold
lysis buffer by gentle inversion and incubate them on the
rotating wheel for 5 min, after which the tubes are again placed
on the magnetic stand to remove the beads and the buffer is
removed. If using colloidal beads (such as those in the Miltenyi
μMACS kit used here), let 500 μL lysis buffer flow through the
column by gravity. In all cases, the buffer should be removed
from the beads after the last wash.
5. If using non-colloidal beads, add Trizol to the tube containing
the beads. If using colloidal beads, remove the column from
the stand and pass the Trizol through the magnetic column
into a 1.5 mL tube, gently pushing with a Pasteur pipette if
necessary.
6. If colloidal beads are used for pull-down, some may be carried
over into the final purified RNA suspension. In our experience,
these beads did not interfere with the further analysis.
7. All material used for electrophoresis and transfer should be
clean or treated with soap, rinsed, and cleaned with 70% EtOH.
8. Electrophoresis should continue until the blue dye migrated to
the middle of the gel. Optionally, a dsRNA ladder such as the
Phi6 dsRNA ladder [7] can be used to monitor dsRNA sizes
and their separation during electrophoresis.
9. The membrane must not dry.
Pull-Down of Viral Double-Stranded RNA
325