7. Filter the supernatant of 16,000 Â g (SN-16) through Miracloth and transfer to 50 mL or a 15 mL plastic conical tube. It is
recommended to centrifuge the samples again in the same
manner to ensure removal of any insoluble material. Keep the
SN-16 on ice before binding of polysomes.
8. Save a small aliquot (200–400 μL) of each (SN-16) to isolate
total RNA. Optionally save other aliquots for western blot
analysis (see Note 8). Keep the rest on ice for affinity purification of polysomes.
Steps 2–8 take approximately 3 h for four 5 mL of tissue
sample. A larger amount of tissue requires more time at the homogenization step.
3.2 Preparation of
the α-FLAGCoupled Beads
These steps can be done prior to the preparation of tissue extracts
described above and will take approximately 1 h and 20 min.
1. Resuspend Protein G Dynabeads by gently shaking the stock
container from side to side by hand. Transfer 50 μL of the
beads per sample (up to 8 samples per batch) to a new 1.5 mL
microfuge tube.
2. Separate the beads by placing to a magnet rack for 3 min.
3. Discard the supernatant by pipetting.
4. Wash beads by adding 400 μL of BWBB and pipet to mix until
complete homogenization is achieved. Repeat steps 2 and 3 to
discard wash.
5. Add the original volume of BWBB per sample (i.e., 200 μL for
4 samples) and prepare 50 μL aliquots in new 1.5 mL microfuge tubes.
6. Place 5 μL (1 μg/μL) of ANTI-FLAG monoclonal antibody
per sample into a new 15 mL conical tube. Dilute with 400 μL
of BWBB per sample.
7. Remove BWBB of the aliquots prepared in step 5 by repeating
steps 2 and 3. Pay attention to avoid bead drying.
8. Add 400 μL of diluted antibody to each aliquot of washed
beads.
9. Incubate with gentle back-and-forth rocking for 1 h at room
temperature.
10. Separate the beads and discard the supernatant by pipetting
carefully.
11. Wash the beads as described in step 4. These are now the
α-FLAG-coupled beads.
12. Add 400 μL of BWBB per sample and precool the beads on ice
until the SN-16 is ready.
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Soledad Traubenik et al.
recommended to centrifuge the samples again in the same
manner to ensure removal of any insoluble material. Keep the
SN-16 on ice before binding of polysomes.
8. Save a small aliquot (200–400 μL) of each (SN-16) to isolate
total RNA. Optionally save other aliquots for western blot
analysis (see Note 8). Keep the rest on ice for affinity purification of polysomes.
Steps 2–8 take approximately 3 h for four 5 mL of tissue
sample. A larger amount of tissue requires more time at the homogenization step.
3.2 Preparation of
the α-FLAGCoupled Beads
These steps can be done prior to the preparation of tissue extracts
described above and will take approximately 1 h and 20 min.
1. Resuspend Protein G Dynabeads by gently shaking the stock
container from side to side by hand. Transfer 50 μL of the
beads per sample (up to 8 samples per batch) to a new 1.5 mL
microfuge tube.
2. Separate the beads by placing to a magnet rack for 3 min.
3. Discard the supernatant by pipetting.
4. Wash beads by adding 400 μL of BWBB and pipet to mix until
complete homogenization is achieved. Repeat steps 2 and 3 to
discard wash.
5. Add the original volume of BWBB per sample (i.e., 200 μL for
4 samples) and prepare 50 μL aliquots in new 1.5 mL microfuge tubes.
6. Place 5 μL (1 μg/μL) of ANTI-FLAG monoclonal antibody
per sample into a new 15 mL conical tube. Dilute with 400 μL
of BWBB per sample.
7. Remove BWBB of the aliquots prepared in step 5 by repeating
steps 2 and 3. Pay attention to avoid bead drying.
8. Add 400 μL of diluted antibody to each aliquot of washed
beads.
9. Incubate with gentle back-and-forth rocking for 1 h at room
temperature.
10. Separate the beads and discard the supernatant by pipetting
carefully.
11. Wash the beads as described in step 4. These are now the
α-FLAG-coupled beads.
12. Add 400 μL of BWBB per sample and precool the beads on ice
until the SN-16 is ready.
460
Soledad Traubenik et al.