5. Commercial magnetic racks can be replaced by placing two
strong neodymium bars on opposite sides of two plastic racks.
For pictures see https://bio-protocol.org/e2458.
6. Use finely grinded tissue to maximize polysome yield. Tissue
collected may be whole plant, a specific organ, or a dissected
region (i.e., root tips). A homogenous pulverization is accomplished with a porcelain mortar and pestle. This step takes
approximately 30 min for four samples. The tissue can be
kept at À80
C until use. Alternatively, it is possible to pulverize
tissue with a frozen bead beater (TissueLyser II, QIAGEN),
when using small amounts of tissue (~100–200 μL). Transgenic tissue from mice and zebrafish embryos is obtained fresh
and immediately homogenized in cold polysome-stabilizing
buffer [32–34, 37]. Drosophila larva dissections can be flashfrozen in dry ice before polysome extraction [35, 36]. Keeping
everything at 4
C throughout the procedure is essential to
maintain the polysome integrity.
7. The amount of tissue depends on the quantity of cells expressing the tagged ribosomal protein (i.e., FLAG-RPL18). In the
case of strong plant promoters, such as CaMV35S, 1 mL of
packed pulverized tissue is enough to get RNA for library
preparation. For larger amount of tissue increase the volume
of PEB buffer to five volumes to facilitate the homogenization.
8. It is recommended to test the efficiency of the procedure by
Western blot with an anti-FLAG antibody conjugated to horseradish peroxidase or an antibody against the epitope tag used
for the ribosomal protein. A band of the expected molecular
weight (e.g., 25 kDa for FLAG-RPL18) should be visualized in
the samples from clarified cellular extract and the eluted material (or the beads after affinity purification, but not in those
corresponding to the unbound fraction as shown in Fig. 3b). A
low amount of tagged ribosomal protein in the affinity-purified
sample may indicate inefficient binding to the beads or low
efficiency of elution. Possible solutions include increasing binding time and elution time, as well as using a fresh stock of beads
and 3X FLAG peptide. It can also be useful to increase the
amount of both of them if FLAG-RPL18 is detected in the
unbound fraction.
9. The polysome composition obtained by TRAP can be evaluated by fractionation in sucrose density gradients. Stable polysome complexes can be eluted by competition incubating for
30 min at 4
C, with back-and-forth shaking, in wash buffer
supplemented with purified peptide (i.e., ~300 ng/mL 3X
FLAG peptide). The eluate should be used immediately for
polysome fractionation in sucrose density gradients. This
468
Soledad Traubenik et al.
strong neodymium bars on opposite sides of two plastic racks.
For pictures see https://bio-protocol.org/e2458.
6. Use finely grinded tissue to maximize polysome yield. Tissue
collected may be whole plant, a specific organ, or a dissected
region (i.e., root tips). A homogenous pulverization is accomplished with a porcelain mortar and pestle. This step takes
approximately 30 min for four samples. The tissue can be
kept at À80
C until use. Alternatively, it is possible to pulverize
tissue with a frozen bead beater (TissueLyser II, QIAGEN),
when using small amounts of tissue (~100–200 μL). Transgenic tissue from mice and zebrafish embryos is obtained fresh
and immediately homogenized in cold polysome-stabilizing
buffer [32–34, 37]. Drosophila larva dissections can be flashfrozen in dry ice before polysome extraction [35, 36]. Keeping
everything at 4
C throughout the procedure is essential to
maintain the polysome integrity.
7. The amount of tissue depends on the quantity of cells expressing the tagged ribosomal protein (i.e., FLAG-RPL18). In the
case of strong plant promoters, such as CaMV35S, 1 mL of
packed pulverized tissue is enough to get RNA for library
preparation. For larger amount of tissue increase the volume
of PEB buffer to five volumes to facilitate the homogenization.
8. It is recommended to test the efficiency of the procedure by
Western blot with an anti-FLAG antibody conjugated to horseradish peroxidase or an antibody against the epitope tag used
for the ribosomal protein. A band of the expected molecular
weight (e.g., 25 kDa for FLAG-RPL18) should be visualized in
the samples from clarified cellular extract and the eluted material (or the beads after affinity purification, but not in those
corresponding to the unbound fraction as shown in Fig. 3b). A
low amount of tagged ribosomal protein in the affinity-purified
sample may indicate inefficient binding to the beads or low
efficiency of elution. Possible solutions include increasing binding time and elution time, as well as using a fresh stock of beads
and 3X FLAG peptide. It can also be useful to increase the
amount of both of them if FLAG-RPL18 is detected in the
unbound fraction.
9. The polysome composition obtained by TRAP can be evaluated by fractionation in sucrose density gradients. Stable polysome complexes can be eluted by competition incubating for
30 min at 4
C, with back-and-forth shaking, in wash buffer
supplemented with purified peptide (i.e., ~300 ng/mL 3X
FLAG peptide). The eluate should be used immediately for
polysome fractionation in sucrose density gradients. This
468
Soledad Traubenik et al.