6
DTT (freshly added), and 2 mM ethylenediaminetetraacetic
acid (EDTA) (filter-sterilized).- Eukaryote lysis buffer: 50 mM HEPES at pH 7.4, 150 mM
NaCl, 1 % NP-40 (see Note 3). - Chelating Sepharose Fast Flow resin (GE Healthcare Life
Science). - Chloramphenicol solution: 34 mg/mL in ethanol (filter to
remove insoluble material if any). - Competent BL21(DE3) pLysS E. coli (EMD Millipore, for-
merly Novagen). - 1 M dithiothreitol (DTT) in water (filter-sterilized).
- Elution buffer: 50 mM Tris at pH 8.0, 0.1 M NaCl, and 0.2 M
imidazole (filter- sterilized) (see Note 4). - Guanidine buffer: 50 mM Tris at pH 8.0, and 6 M guanidine
hydrochloride. - 1.2× initiator caspase buffer: 60 mM 4-(2-hydroxyethyl)piper-
azine-1-ethanesulfonic acid (HEPES) at pH 7.4 (NaOH),
1.2 M sodium citrate, 60 mM NaCl, 0.012 % w/v CHAPS,
and 12 mM DTT (freshly added) (filter-sterilized). - Isopropyl β-d-1-thiogalactopyranoside (IPTG; keep as powder
at −20 °C). - Kanamycin solution: 20 mg/mL in water (filter-sterilized).
- LB agar plates (1 L): 10 g tryptone, 5 g Bacto yeast extract, 5 g
NaCl, 15 g agar (autoclave-sterilized); 100 μg/mL ampicillin
or 20 μg/mL kanamycin, and 25 μg/mL chloramphenicol
(see Note 5 and Subheading 3.1.1.1 for antibiotic selection). - Bacterial lysis buffer: 50 mM Tris at pH 8.0 and 0.1 M NaCl
(autoclave-sterilized).
0.1 M NiSO 4 solution in water (filter-sterilized). - PBS: 10.2 mM Na 2 HPO 4 , 1.76 mM KH 2 PO 4 at pH 7.4,
137 mM NaCl, and 2.7 mM KCl (autoclave-sterilized). - PBS-EGTA/EDTA: PBS, 1 mM EGTA (ethyleneglycoltet-
raacetic acid), and 1 mM EDTA. - Refolding buffer #1: 55 mM Tris at pH 8.0, 440 mM l-
arginine, 400 mM NaCl, 10 mM DTT, 1 mM EGTA, and
0.88 mM KCl. - Refolding buffer #2: 50 mM HEPES at pH 8.0, 200 mM
NaCl, 10 mM DTT, and 0.2 % Tween 20. - 3× SDS-PAGE gel loading buffer that is suitable for the SDS-
PAGE gel system used. - Urea buffer: 50 mM Tris at pH 8.0, 8 M urea.
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