into a 1.5 mL tube, pipette the labeling mix into the column,
and centrifuge as above. Following this step, the labeled probe
is in the bottom of the tube while the column contains the
unincorporated radioactive nucleotides. Discard the column
and denature the probe by placing it at 95
C for 2 min and
then on ice.
9. Add the probe to the hybridization buffer in the hybridization
tube containing the membrane. Return the tube to the hybridization oven and incubate under rotation at 42
C for at least
4 h.
10. Open the tube and discard the hybridization buffer containing
the labeled probe (see Note 11).
11. Add 20 mL wash buffer and incubate the membrane for
10–15 min in the hybridization oven at 50
C with rotation.
12. Discard the wash buffer.
13. Repeat steps 11 and 12 two more times.
14. Use a pair of tweezers to remove the membrane from the
hybridization buffer and place it to dry on a paper towel.
Once dry, place the membrane in a plastic envelope, and then
place the envelope into a radioactivity-detection instrument of
choice (cassette and autoradiographic film, or phosphorimager screen and detector) (Fig. 3b).
3.5 Western Blot
The presence of proteins in the pull-down fraction should be
verified before these are analyzed by mass spectrometry. Since B2:
GFP is the bait protein through which dsRNA was pulled down, at
least the presence of B2:GFP should be confirmed. Furthermore,
the amount of B2:GFP should be confirmed as being (i) consistent
between technical replicates and (ii) comparable to the amount of
GFP pulled down in control samples. This quality control can be
easily carried out by performing Western blot analysis of the bait
GFP/B2:GFP proteins in the input and IPed (bead-bound) fractions using anti-GFP antibodies (Fig. 4) (see Note 12).
1. For samples to use for mass spectrometry, from pull-downs
performed in triplicate, resuspend the magnetic beads in a
solution consisting of three parts protein resuspension buffer
and one part protein-loading buffer, preheated to 95
C (see
Note 5, but using the buffer instead of TRIzol). In parallel,
add a ¼ volume of protein-loading buffer to the input and
flow-through fractions collected during the pull-down experiment and vortex. Denature all samples by heating them for
2–3 min to 95
C and then placing them for 5 min on ice.
Samples can now be stored at À20
C.
2. To provide a complete analysis of the pull-down experiment
used for northern and north-western blot (see Subheadings 3.3
Pull-Down of Viral Double-Stranded RNA
319
and centrifuge as above. Following this step, the labeled probe
is in the bottom of the tube while the column contains the
unincorporated radioactive nucleotides. Discard the column
and denature the probe by placing it at 95
C for 2 min and
then on ice.
9. Add the probe to the hybridization buffer in the hybridization
tube containing the membrane. Return the tube to the hybridization oven and incubate under rotation at 42
C for at least
4 h.
10. Open the tube and discard the hybridization buffer containing
the labeled probe (see Note 11).
11. Add 20 mL wash buffer and incubate the membrane for
10–15 min in the hybridization oven at 50
C with rotation.
12. Discard the wash buffer.
13. Repeat steps 11 and 12 two more times.
14. Use a pair of tweezers to remove the membrane from the
hybridization buffer and place it to dry on a paper towel.
Once dry, place the membrane in a plastic envelope, and then
place the envelope into a radioactivity-detection instrument of
choice (cassette and autoradiographic film, or phosphorimager screen and detector) (Fig. 3b).
3.5 Western Blot
The presence of proteins in the pull-down fraction should be
verified before these are analyzed by mass spectrometry. Since B2:
GFP is the bait protein through which dsRNA was pulled down, at
least the presence of B2:GFP should be confirmed. Furthermore,
the amount of B2:GFP should be confirmed as being (i) consistent
between technical replicates and (ii) comparable to the amount of
GFP pulled down in control samples. This quality control can be
easily carried out by performing Western blot analysis of the bait
GFP/B2:GFP proteins in the input and IPed (bead-bound) fractions using anti-GFP antibodies (Fig. 4) (see Note 12).
1. For samples to use for mass spectrometry, from pull-downs
performed in triplicate, resuspend the magnetic beads in a
solution consisting of three parts protein resuspension buffer
and one part protein-loading buffer, preheated to 95
C (see
Note 5, but using the buffer instead of TRIzol). In parallel,
add a ¼ volume of protein-loading buffer to the input and
flow-through fractions collected during the pull-down experiment and vortex. Denature all samples by heating them for
2–3 min to 95
C and then placing them for 5 min on ice.
Samples can now be stored at À20
C.
2. To provide a complete analysis of the pull-down experiment
used for northern and north-western blot (see Subheadings 3.3
Pull-Down of Viral Double-Stranded RNA
319