3.9 Radioactive
Labeling of RNA
1. Mix beads thoroughly and transfer 200 μL (20%) of the second
wash including beads to a new 1.5 mL LoBind
® tube.
2. Keep the remaining 800 μL (80%) of the second wash including
beads and store on ice until step 11.
3. Centrifuge for 1 min at 500 Â g and discard supernatant.
4. Resuspend beads in 18 μL PNK mix (2 μL 10Â buffer A,
14.5 μL H 2 O, 1 μL T4-PNK, 0.5 μL RiboLock).
5. Add 2 μL [γ32
P] ATP.
6. Incubate for 20 min at 37
C and 1100 rpm in a thermomixer.
7. Wash three times with 800 μL of ice-cold PNK wash buffer
(pH 7.4) for 1 min under constant agitation.
8. After each washing step, centrifuge for 1 min at 500 Â g and
discard supernatant.
9. Bring 4Â NuPAGE™ LDS sample buffer to room temperature
and dilute it to 2Â with ddH 2 O.
10. Add 20 μL of 2Â NuPAGE™ LDS sample buffer to the beads
and mix carefully by pipetting (see Note 11).
11. Remove supernatant from the remaining cold beads from
step 2 and add the 20% radioactively labeled beads to the
80% cold beads.
12. Incubate at 95
C for 10 min.
13. Place tube on ice and centrifuge briefly to precipitate the beads.
14. Collect the supernatant and store it on ice until loading of
the gel.
3.10 SDS-PAGE
1. Prepare 1Â MOPS SDS running buffer by diluting 20Â stock
solution with ddH 2 O.
2. Set up a precast 4–12% NuPAGE Bis-Tris gel according to the
manufacturer’s instructions.
3. Load the supernatant of the beads from Subheading 3.9,
step 14, on the gel (see Note 12).
4. Load 5 μL of prestained protein ladder.
5. Run the gel for 60 min at 180 V in 1Â MOPS SDS running
buffer according to the manufacturer’s instructions.
6. Remove the gel front (~0.5 cm) and discard as solid radioactive
waste (contains free radioactive [γ32 P] ATP).
3.11 Blot and
Isolation of RNAProtein Complexes
1. Set up the XCell II Blot Module according to the manufacturer’s instructions.
2. Prepare transfer buffer (see Subheading 2.11) by freshly adding
10% methanol.
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263
Labeling of RNA
1. Mix beads thoroughly and transfer 200 μL (20%) of the second
wash including beads to a new 1.5 mL LoBind
® tube.
2. Keep the remaining 800 μL (80%) of the second wash including
beads and store on ice until step 11.
3. Centrifuge for 1 min at 500 Â g and discard supernatant.
4. Resuspend beads in 18 μL PNK mix (2 μL 10Â buffer A,
14.5 μL H 2 O, 1 μL T4-PNK, 0.5 μL RiboLock).
5. Add 2 μL [γ32
P] ATP.
6. Incubate for 20 min at 37
C and 1100 rpm in a thermomixer.
7. Wash three times with 800 μL of ice-cold PNK wash buffer
(pH 7.4) for 1 min under constant agitation.
8. After each washing step, centrifuge for 1 min at 500 Â g and
discard supernatant.
9. Bring 4Â NuPAGE™ LDS sample buffer to room temperature
and dilute it to 2Â with ddH 2 O.
10. Add 20 μL of 2Â NuPAGE™ LDS sample buffer to the beads
and mix carefully by pipetting (see Note 11).
11. Remove supernatant from the remaining cold beads from
step 2 and add the 20% radioactively labeled beads to the
80% cold beads.
12. Incubate at 95
C for 10 min.
13. Place tube on ice and centrifuge briefly to precipitate the beads.
14. Collect the supernatant and store it on ice until loading of
the gel.
3.10 SDS-PAGE
1. Prepare 1Â MOPS SDS running buffer by diluting 20Â stock
solution with ddH 2 O.
2. Set up a precast 4–12% NuPAGE Bis-Tris gel according to the
manufacturer’s instructions.
3. Load the supernatant of the beads from Subheading 3.9,
step 14, on the gel (see Note 12).
4. Load 5 μL of prestained protein ladder.
5. Run the gel for 60 min at 180 V in 1Â MOPS SDS running
buffer according to the manufacturer’s instructions.
6. Remove the gel front (~0.5 cm) and discard as solid radioactive
waste (contains free radioactive [γ32 P] ATP).
3.11 Blot and
Isolation of RNAProtein Complexes
1. Set up the XCell II Blot Module according to the manufacturer’s instructions.
2. Prepare transfer buffer (see Subheading 2.11) by freshly adding
10% methanol.
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263