5occur in E. coli, and caspases do not require any posttranslational
modification to display full activity. Consequently, it is relatively
easy to obtain enzymatically pure caspase preparations from E. coli.2 Materials
- 15-mL bacterial culture tubes.
- 1-L baffled culture flasks.
- 250-mL baffled culture flask.
- Benchtop microcentrifuge.
- Benchtop centrifuge for 15/50-mL conical tubes.
- 15-mL conical tubes.
- 10,000 MWCO dialysis tube (Spectrum Laboratories or
equivalent). - 0.45-μm 150-mL Durapore Stericup™ HV (Millipore) or
equivalent. - Econo-Pac 0.7 × 5.0-cm column (Bio-Rad) or equivalent.
- Floor centrifuge with 8 × 50 mL (Sorvall SW-34 or equivalent)
and 6 × 250 mL (Sorvall SLA-1500 or equivalent) or higher
volume capacity rotor. - 1.5-mL microfuge tubes.
- Multichannel pipettors (8 channels, 200 and 10 μL).
- 50/100-mL plastic beaker.
- Repeating pipettor with various volume tips (20–200 μL).
- Shaking incubator.
- Spectrophotometer.
- 10,000 MWCO spin concentrator (Millipore or equivalent).
- Thermostatic fluorescence plate reader for 96-well plates.
- Ultrasonic cell disruptor equipped with a large probe.
- 96-well plates, preferentially black (see Note 1).
- 7-Amino-3-trifluoromethylcoumarin (Afc) 10 mM in dimethyl
sulfoxide (DMSO; keep at −20 °C). See Subheading 3.2.1.1 for
the preparation of the Afc standard solution. - Afc-based fluorogenic peptidic substrates, such as Ac-DEVD-
Afc: 20 mM in DMSO (keep at −20 °C) (see Note 2). - Ampicillin solution: 100 mg/mL in water (filter-sterilized).
- 2× executioner caspase buffer: 20 mM 1,4-piperazinediethane-
sulfonic acid (PIPES) at pH 7.2 (NaOH), 200 mM NaCl,
20 % w/v sucrose, 0.2 % w/v 3-[(3- cholamidopropyl)
dimethylammonio]-1-propanesulfonate (CHAPS), 20 mM
2.1 Equipment
2.2 Reagents
Apoptotic Caspases Assays