10. Electrophoresis should continue until the blue dye migrated to
the middle of the gel.
11. The hybridization buffer containing the labeled probe can be
appropriately stored and used again, taking into account the
radioactive decay.
12. Optionally, western blot analysis can include the “flowthrough” samples collected during the pull-down experiment.
Western blot analysis of “input” and “flow-through” samples
on the same gel/membrane allows to assess the pull-down
efficiency by comparing the amount of B2:GFP present in the
lysate before and after the IP. For an example of such an
analysis see ref. 8.
13. Antibody dilution and time of incubation depend on the antibody used. For the study in ref. 8, a rabbit polyclonal antibody
raised against GFP was used. The antibody was applied in
1:30,000 to 1:60,000 dilution and used for incubation overnight. If using commercially available monoclonal antibodies,
follow the instructions of the manufacturer.
14. Incubation with a secondary antibody is not necessary if the
primary GFP antibody is coupled to horseradish peroxidase.
15. If clones for agroinfection are not available, infection should be
carried out by rub inoculation with a crude extract of infected
tissue or with purified virus. This should be performed 24 h
after agro-infiltration of the plasmid encoding the protein
candidate of interest. Prepare the viral inoculum appropriately
according to the virus, sprinkle celite/carborundum on
the adaxial side of the agroinfiltrated leaves, and gently rub
the viral inoculum with a gloved finger. After 10–15 min, rinse
the leaves with water and return the plants to the growth
chamber/greenhouse. Remember also to use some of the
plants as noninfected/mock-treated controls.
16. In case the virus was delivered by rub inoculation, it is advisable
to perform the observation at 3 days after virus infection. This
time is needed for infection foci to form.
17. Virus infection may interfere with the expression of the
dsRNA-binding protein candidate. This was observed for
many candidates that were tested in ref. 8. While we do not
know the reason for this phenomenon, a possible explanation is
that infected tissues undergo a process similar to the previously
described host gene shutoff [10]. In our hands, this phenomenon worsened with the time after virus infection. Therefore, it
is advisable to evaluate the leaf disks as soon as possible after the
appearance of the first infection foci.
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Marco Incarbone and Christophe Ritzenthaler
the middle of the gel.
11. The hybridization buffer containing the labeled probe can be
appropriately stored and used again, taking into account the
radioactive decay.
12. Optionally, western blot analysis can include the “flowthrough” samples collected during the pull-down experiment.
Western blot analysis of “input” and “flow-through” samples
on the same gel/membrane allows to assess the pull-down
efficiency by comparing the amount of B2:GFP present in the
lysate before and after the IP. For an example of such an
analysis see ref. 8.
13. Antibody dilution and time of incubation depend on the antibody used. For the study in ref. 8, a rabbit polyclonal antibody
raised against GFP was used. The antibody was applied in
1:30,000 to 1:60,000 dilution and used for incubation overnight. If using commercially available monoclonal antibodies,
follow the instructions of the manufacturer.
14. Incubation with a secondary antibody is not necessary if the
primary GFP antibody is coupled to horseradish peroxidase.
15. If clones for agroinfection are not available, infection should be
carried out by rub inoculation with a crude extract of infected
tissue or with purified virus. This should be performed 24 h
after agro-infiltration of the plasmid encoding the protein
candidate of interest. Prepare the viral inoculum appropriately
according to the virus, sprinkle celite/carborundum on
the adaxial side of the agroinfiltrated leaves, and gently rub
the viral inoculum with a gloved finger. After 10–15 min, rinse
the leaves with water and return the plants to the growth
chamber/greenhouse. Remember also to use some of the
plants as noninfected/mock-treated controls.
16. In case the virus was delivered by rub inoculation, it is advisable
to perform the observation at 3 days after virus infection. This
time is needed for infection foci to form.
17. Virus infection may interfere with the expression of the
dsRNA-binding protein candidate. This was observed for
many candidates that were tested in ref. 8. While we do not
know the reason for this phenomenon, a possible explanation is
that infected tissues undergo a process similar to the previously
described host gene shutoff [10]. In our hands, this phenomenon worsened with the time after virus infection. Therefore, it
is advisable to evaluate the leaf disks as soon as possible after the
appearance of the first infection foci.
326
Marco Incarbone and Christophe Ritzenthaler