75Biosciences) supplemented with 10 mM 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES), 100 mM penicillin/
streptomycin (CSL Biosciences), and 10 % fetal bovine serum.- Trypsin (0.25 %) diluted in Hank’s buffered salt solution
(HBSS) (SAFC Biosciences). This can be aliquoted and stored
at −20 °C. - Phosphate buffered saline (PBS): 2 mM KH 2 PO 4 , 10 mM
Na 2 HPO 4 , 150 mM NaCl, pH 7.2. - Water bath at 37 °C.
- Laminar fl ow hood.
- Bench top centrifuge with swing-bucket rotor.
- Cell extraction buffer: 50 mM HEPES-KOH pH 7.5, 50 mM
NaCl, 10 mM dithiothreitol (DTT), 0.5 mM ethylenediami-
netetraacetic acid (EDTA), 0.1 % 3-[(3-cholamidopropyl)-
dimethylammonio]-1-propanesulfonate (CHAPS), 10 %
sucrose, pH 7.5, and containing protease inhibitor cocktail
such as Complete™ (Roche). - 2× Protein Loading Buffer (PLB): 100 mM Tris–HCl pH 6.8,
200 mM DTT, 20 % glycerol, 4 % SDS, 0.2 % bromophenol blue. - Liquid nitrogen.
- Ice-cold water.
- Water bath (with boiling water).
- Bench top centrifuge.
- Protein concentration assay reagent (such as BCA or Bradford
reagent). - Plate reader equipped to measure absorbance between 540
and 595 nm. - Competent Escherichia coli bacterial cells optimized for expres-
sion of recombinant proteins (such as BL21 (DE3) cells or
BL21 star cells). - Luria broth (LB): 1 % Bacto-tryptone, 0.5 % Bacto-yeast
extract, 1 % NaCl, pH 7.0. - 1 mM isopropyl β- D -thiogalactoside (IPTG).
- Caspase-2 bacterial expression constructs (e.g., pET- Caspase-2
or pGEX-Caspase-2). - Antibiotics for selective growth of transformed bacteria (e.g.,
100 μg/ml ampicillin; 34 μg/ml chloramphenicol). - Petri dishes (90 mm) containing LB/agar: 1 % Bacto-tryptone,
0.5 % Bacto-yeast extract, 1 % NaCl pH 7.0, 1.5 % Bacto-agar. - Orbital shaking incubator at 30 °C.
- Orbital shaker at 4 °C.
2.2 Protein
Extraction
2.3 Preparation
of Recombinant
Caspase-2
Assays for Studying Caspase-2