4. 1 M MgCl 2 .
5. 20% (v/v) Detergent mix: Dissolve 4 g Brij
® L23 Polyoxyethylene (23) lauryl ether (Brij 35) by heating to about 60
C in
4 mL of TritonX-100, 4 mL of Igepal CA-630, and 4 mL of
Tween-20 (polyoxyethylene (20) sorbitan monolaurate), and
fill up with molecular biology-grade water to 20 mL (see
Note 4).
6. 20% (v/v) Polyoxyethylene (10) tridecyl ether (PTE) in molecular biology-grade water: Shake the stock solution before
pipetting PTE. Protect from light.
7. 10% (v/v) Tween-20 in molecular biology-grade water.
8. 5 M NaCl.
9. 40% (v/v) Poly(ethylene glycol) 8000 (PEG8000) in molecular biology-grade water.
The following solutions are not autoclaved and are stored in
aliquots at À20
C.
10. 0.5 M Dithiothreitol (DTT) (see Note 2).
11. 100 mM Phenylmethylsulfonyl fluoride (PMSF): Dissolve
87 mg of PMSF in 3.5 mL of isopropanol. Fill up with isopropanol to 5 mL (see Note 2).
12. 50 mg/mL Cycloheximide: Dissolve 50 mg of cycloheximide
in 1 mL of 96% (v/v) ethanol (see Note 2). Freshly prepared in
each occasion.
13. 50 mg/mL Chloramphenicol: Dissolve 50 mg of chloramphenicol in 1 mL of 96% (v/v) ethanol (see Note 2).
2.3 Buffers for TRAP
The following buffers are prepared fresh on the day of use from
stock solutions (described in the previous Subheading 2.2) and
kept on ice for the duration of the procedure.
1. Polysome extraction buffer (PEB): 200 mM Tris–HCl,
200 mM KCl, 25 mM EGTA, 35 mM MgCl 2 , 1% (v/v) detergent mix, 1% (v/v) PTE, 1 mM DTT, 1 mM PMSF, 50 μg/μL
cycloheximide, and 50 μg/μL chloramphenicol. Keep the
buffer on ice for 10 min before adding DTT, PMSF, cycloheximide, and chloramphenicol (see Note 4).
2. Bead wash and binding buffer (BWBB): 200 mM Tris–HCl,
200 mM KCl, 25 mM EGTA, 35 mM MgCl 2 , 0.02% (v/v)
Tween-20.
3. Wash buffer (WB): 200 mM Tris–HCl, 200 mM KCl, 25 mM
EGTA, 35 mM MgCl 2 , 1 mM DTT, 1 mM PMSF, 50 μg/μL
cycloheximide, and 50 μg/μL chloramphenicol. Keep the
buffer on ice for 10 min before adding DTT, PMSF, cycloheximide, and chloramphenicol.
458
Soledad Traubenik et al.
5. 20% (v/v) Detergent mix: Dissolve 4 g Brij
® L23 Polyoxyethylene (23) lauryl ether (Brij 35) by heating to about 60
C in
4 mL of TritonX-100, 4 mL of Igepal CA-630, and 4 mL of
Tween-20 (polyoxyethylene (20) sorbitan monolaurate), and
fill up with molecular biology-grade water to 20 mL (see
Note 4).
6. 20% (v/v) Polyoxyethylene (10) tridecyl ether (PTE) in molecular biology-grade water: Shake the stock solution before
pipetting PTE. Protect from light.
7. 10% (v/v) Tween-20 in molecular biology-grade water.
8. 5 M NaCl.
9. 40% (v/v) Poly(ethylene glycol) 8000 (PEG8000) in molecular biology-grade water.
The following solutions are not autoclaved and are stored in
aliquots at À20
C.
10. 0.5 M Dithiothreitol (DTT) (see Note 2).
11. 100 mM Phenylmethylsulfonyl fluoride (PMSF): Dissolve
87 mg of PMSF in 3.5 mL of isopropanol. Fill up with isopropanol to 5 mL (see Note 2).
12. 50 mg/mL Cycloheximide: Dissolve 50 mg of cycloheximide
in 1 mL of 96% (v/v) ethanol (see Note 2). Freshly prepared in
each occasion.
13. 50 mg/mL Chloramphenicol: Dissolve 50 mg of chloramphenicol in 1 mL of 96% (v/v) ethanol (see Note 2).
2.3 Buffers for TRAP
The following buffers are prepared fresh on the day of use from
stock solutions (described in the previous Subheading 2.2) and
kept on ice for the duration of the procedure.
1. Polysome extraction buffer (PEB): 200 mM Tris–HCl,
200 mM KCl, 25 mM EGTA, 35 mM MgCl 2 , 1% (v/v) detergent mix, 1% (v/v) PTE, 1 mM DTT, 1 mM PMSF, 50 μg/μL
cycloheximide, and 50 μg/μL chloramphenicol. Keep the
buffer on ice for 10 min before adding DTT, PMSF, cycloheximide, and chloramphenicol (see Note 4).
2. Bead wash and binding buffer (BWBB): 200 mM Tris–HCl,
200 mM KCl, 25 mM EGTA, 35 mM MgCl 2 , 0.02% (v/v)
Tween-20.
3. Wash buffer (WB): 200 mM Tris–HCl, 200 mM KCl, 25 mM
EGTA, 35 mM MgCl 2 , 1 mM DTT, 1 mM PMSF, 50 μg/μL
cycloheximide, and 50 μg/μL chloramphenicol. Keep the
buffer on ice for 10 min before adding DTT, PMSF, cycloheximide, and chloramphenicol.
458
Soledad Traubenik et al.