Protein Protocols Protein Protocols

160 Bizios
2. Materials
2.1. Equipment
1. 0.2 µm Filters.
2. Heat block or 100°C water bath.
2.2. Reagents
1. Complete culture medium: 90% RPMI-1640, 10% fetal bovine serum (FBS), streptomycin–penicillin:
Mix 990 mL of RPMI-1640 with 100 mL of FBS, and add 10 mL of streptomycin–penicillin
(100×). Cold-filter sterilize using a 0.2 µm filter. Store at 4°C. Maintain
the Jurkat T lymphoblasts at a concentration of 105 –106 cells/mL (see Note 2).
2. Methionine-free medium: 90% Methionine-free RPMI-1640, 10% dialyzed FBS (dFBS):
Mix 990 mL of methionine-free RPMI-1640 with 100 mL of dFBS. Cold-filter sterilize
using a 0.2µm filter. Store at 4°C.
3. Sodium phosphate-free medium: 90% sodium phosphate-free RPMI-1640, 10% FBS: Mix
90% sodium phosphate free RPMI with 10% FBS. Cold-filter-sterilize Store at 4°C.
4. 35S-label (EXPRE35S35S[ 35S] methionine/cysteine mix (New England Nuclear [NEN]).
5. 32P orthophospa hte (NEN).
6. Phosphate-buffered saline (PBS): Mix 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and
0.24 g of KH2PO4 in 800 mL of dH20, and adjust the pH to 7.4 with HCl. Add dH2O to a final volume of 1000 mL and autoclave. Store at room temperature.
7. Dilute SDS (dSDS): 0.3% SDS, 1% β-mercaptoethanol (β-ME), 0.05 M Tris-HCl,pH 8.0.
In a cold room, mix 3.0 g of SDS, 4.44 g of Tris-HCl, 2.65 g Tris base, and10 mL of β-ME
in distilled water, and adjust the final volume to 1 L with dH2O. Aliquot 500 mL into
microcentrifuge tubes, and store at –70°C.
8. DNase/RNase solution: 1 mg/mL of DNase I, 0.5 mg/mL of RNase A, 0.5 M Tris-HCl,
0.05 M MgCl2, pH 7.0. Thaw RNase, Tris, and MgCl2 stocks, and thoroughly mix 2.5 mg
of RNase A (Worthington Enzymes), 1585 µL of 1.5 M Tris-HCl, 80 µL of 1.5 M Tris base,
250 µL of 1.0 M MgCl2, and 2960 µL of dH2O. Mix the liquids and 5 mg of DNase I
(Worthington Enzymes). Do not filter. Keep cool while dispensing into microcentrifuge
tubes. Make 50 µL aliquots, and store at –70°C.
9. 1.5 M Tris-HCl solution: Weigh out 11.8 g of desiccated Tris-HCl, and add 41.4 g of
dH2O. Mix well and filter through a 0.2-µm filter. Aliquot into microcentrifuge
tubes, and store at –70°C.
10. 1.5 M Tris base solution: Weigh out 9.09 g of Tris base, and add 41.4 g of dH2O. Mix and
filter through a 0.2-µm filter. Aliquot into microcentrifuge tubes, and store at –70°C.
11. 1.0 M MgCl2 solution: Weigh out 30.3 g of MgCl2, and add 85.9 g of dH2O. Mix and filter
through a 0.2-µm filter. Aliquot into microcentrifuge tubes, and store at –70°C.
12. Sample buffer solution (SB): 9.95 M urea, 4.0% Nonident P-40 (NP40) (Sigma), 2%
pH 6.0–8.0 ampholytes, 100 mM dithiothreitol (DTT). Mix 59.7 g of urea, 44.9 g of dH2O, 4.0 g of NP40, 5.5 g of pH 6.0 –8.0 ampholytes (AP Biotech), 1.54 g of DTT (Calbiochem)
in this order in a 30–37º C waterbath just long enough to dissolve the urea. Filter through
a 0.2-µm filter and aliquot 1 mL into microcentrifuge tubes. Snap-freeze in liquid nitrogen,
and store at –70°C.
13. Sample buffer with SDS solution (SBS): 9.95 M urea, 4.0% NP40, 0.3% SDS, 2%
pH 6.0–8.0 ampholytes, 100 mM DTT. Mix 59.7 g of urea, 44.9 g dH2O, 4.0 g of NP40,
0.3 g of SDS, 5.5 g of pH 6.0–8.0 ampholytes, 1.54 g of DTT in this order in a 30–37°C
waterbath just long enough to dissolve the urea. Filter through a 0.2-µm filter and aliquot
1 mL into microcentrifuge tubes. Snap-freeze in liquid nitrogen, and store at –70°C.