240
- Luria Broth (LB) medium: tryptone 10 g/L, yeast extract
5 g/L, NaCl 10 g/L. Add 15 g/L of Bacto Agar to solidify
the medium, when required. Autoclave for 20 min at 120 °C. - Bacterial strain for IPTG-inducible expression (Stratagene,
230245 or similar). - Sonication buffer: 100 mM Tris–HCl pH 8.0, 300 mM NaCl,
10 mM ethylenediaminetetraacetic acid (EDTA), 100 μM
phenylmethanesulfonyl-fl uoride (PMSF), 20 μM pepstatin A,
10 μM leupeptin. - Binding buffer: 50 mM NaH 2 PO 4 pH 8.0, 300 mM NaCl,
10 mM imidazole, 10 mM β-mercaptoethanol. - Washing buffers for native conditions: 50 mM NaH 2 PO 4
pH 8.0, 10 mM β-mercaptoethanol, 50–100 mM imidazole. - Elution buffer for native conditions: 50 mM NaH 2 PO 4
pH 8.0, 10 mM β-mercaptoethanol, 250 mM imidazole. - Lysis buffer for denaturing conditions: 100 mM NaH 2 PO 4
pH 8.0, 100 mM Tris–HCl, 6 M guanidine–HCl, 100 μM
PMSF, 20 μM pepstatin A, 10 μM leupeptin. Adjust pH to 8.0. - Washing buffers for denaturing conditions: 100 mM NaH 2 PO 4
pH 8.0, 10 mM β-mercaptoethanol, 8 M urea. Prepare a series
of buffers with a pH gradient from pH 8.0 to pH 6.0. - Elution buffer for denaturing conditions: 100 mM NaH 2 PO 4
pH 8.0, 10 mM β-mercaptoethanol, 8 M urea. Adjust pH
to 4.5. - Bradford reagent (Thermo Scientifi c, 23200 or similar).
- Dialysis buffer: 50 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) pH7.5, 150 mM
NaCl, 5 mM dithiothreitol (DTT). - Dialysis tubing or cassette.
- Reaction buffer: 50 mM HEPES (if optimal pH is not known
a range of pH from 4.0 to 9.0 with 0.5 increment should be
used for optimization of the reaction), 0.1 % (w/v)
3-[(3-cholamido- propyl)dimethylammonio]-1-propanesulfo-
nate (CHAPS), 5 mM DTT, 50 mM CaCl 2 (do not add for
negative controls). - 7-Amino-4-methylcoumarin (AMC)-labeled substrate, for
example Boc-Glu(OBzl)-Gly-Arg-AMC-HCl (Bachem I-1545
or similar) or Ac-VRPR-AMC (Bachem I-1965). Make a
10 mM stock solution in DMSO, aliquot, and store at −20 °C. - AMC (Bachem Q-1025 or similar). Make a 100 mM stock
solution in DMSO, aliquot, and store at −20 °C. Before use
dilute additionally with reaction buffer to 24 μM in order to
prepare standard solutions.
2.2 Measurement
of Recombinant
Metacaspase Activity
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