2.2 Labeling
1. 500 mM 4-Thiouridine (4SU). Store at À20
C.
2. 500 mM Biotin-phenol (BP) in DMSO (may need to be sonicated to dissolve, store 50 μL aliquots at À80
C).
3. 1 M Sodium azide: For long term store aliquots at À20
C.
4. Sodium ascorbate (powder, needed fresh).
5. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid) (powder, needed fresh).
6. 30% wt/wt Hydrogen peroxide stock: Needed fresh, do not
prepare diluted solution in advance.
7. Any standard fixative to process cells for imaging by fluorescence microscopy. In our hands 16% PFA freshly diluted to 4%
in PBS performed better than methanol at À20
C.
8. Phosphate-buffered saline (PBS) pH 7.4: 0.144 g/L KH 2 PO 4 ,
9 g/L NaCl, 0.795 g/L Na 2 HPO 4 -7H 2 O.
9. 10Â PBS pH 7.4: 1.44 g/L KH 2 PO 4 , 90 g/L NaCl, 7.95 g/L
Na 2 HPO 4 -7H 2 O.
10. DMSO.
11. UV cross-linker equipped with far-UV light bulbs (wavelength
>310 nm, preferably 365 nm).
12. Sonicator with probe that fits into a microcentrifuge tube.
2.3 Cell Extraction
and Streptavidin
Affinity Purification
1. DEPC-treated double-distilled water (will be referred in the
Methods as water).
2. RIPA extraction buffer: 50 mM Tris–HCl, 150 mM NaCl,
0.1% (wt/vol) SDS, 0.5% (wt/vol) sodium deoxycholate, 1%
(wt/vol) Triton X-100. Adjust pH to 7.5 with HCl. Can be
stored at 4
C for many months.
3. 100 mM PMSF in ethanol.
4. Protease inhibitor cocktail without EDTA.
5. Sodium ascorbate (powder, needed fresh).
6. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (powder, needed fresh).
7. 1 M Sodium azide.
8. Pierce 660 nm protein assay reagent.
9. Ponceau S solution.
10. Streptavidin-coupled horseradish peroxidase.
11. Streptavidin-coupled magnetic beads.
12. Magnetic rack for 1.5 mL tubes.
13. 1 M KCl.
14. 0.1 M Na 2 CO 3 .
15. Urea (powder, needed fresh).
286
Daniel Benhalevy and Markus Hafner
1. 500 mM 4-Thiouridine (4SU). Store at À20
C.
2. 500 mM Biotin-phenol (BP) in DMSO (may need to be sonicated to dissolve, store 50 μL aliquots at À80
C).
3. 1 M Sodium azide: For long term store aliquots at À20
C.
4. Sodium ascorbate (powder, needed fresh).
5. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid) (powder, needed fresh).
6. 30% wt/wt Hydrogen peroxide stock: Needed fresh, do not
prepare diluted solution in advance.
7. Any standard fixative to process cells for imaging by fluorescence microscopy. In our hands 16% PFA freshly diluted to 4%
in PBS performed better than methanol at À20
C.
8. Phosphate-buffered saline (PBS) pH 7.4: 0.144 g/L KH 2 PO 4 ,
9 g/L NaCl, 0.795 g/L Na 2 HPO 4 -7H 2 O.
9. 10Â PBS pH 7.4: 1.44 g/L KH 2 PO 4 , 90 g/L NaCl, 7.95 g/L
Na 2 HPO 4 -7H 2 O.
10. DMSO.
11. UV cross-linker equipped with far-UV light bulbs (wavelength
>310 nm, preferably 365 nm).
12. Sonicator with probe that fits into a microcentrifuge tube.
2.3 Cell Extraction
and Streptavidin
Affinity Purification
1. DEPC-treated double-distilled water (will be referred in the
Methods as water).
2. RIPA extraction buffer: 50 mM Tris–HCl, 150 mM NaCl,
0.1% (wt/vol) SDS, 0.5% (wt/vol) sodium deoxycholate, 1%
(wt/vol) Triton X-100. Adjust pH to 7.5 with HCl. Can be
stored at 4
C for many months.
3. 100 mM PMSF in ethanol.
4. Protease inhibitor cocktail without EDTA.
5. Sodium ascorbate (powder, needed fresh).
6. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (powder, needed fresh).
7. 1 M Sodium azide.
8. Pierce 660 nm protein assay reagent.
9. Ponceau S solution.
10. Streptavidin-coupled horseradish peroxidase.
11. Streptavidin-coupled magnetic beads.
12. Magnetic rack for 1.5 mL tubes.
13. 1 M KCl.
14. 0.1 M Na 2 CO 3 .
15. Urea (powder, needed fresh).
286
Daniel Benhalevy and Markus Hafner