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- Extraction buffer: 0.1 M Tris–HCl (pH 8.0), 0.14 M NaCl,
and 1 mM phenylmethane sulfonyl fl uoride. - 20 mM Tris–HCl (pH 8.0).
- 1 M Tris–HCl (pH 8.0).
- 0–1 M linear NaCl gradient.
- 20 mM acetate buffer (pH 4.5).
- 25 mM ethanolamine (pH 8.3).
- Polybuffer: Polybuffer 74 (70 %) + Polybuffer 96 (30 %), pH
adjusted to 5.0 with acetic acid. - Bio-Rad protein assay kit or similar.
- Coomassie Brilliant Blue (CBB).
- H99 antibody directed to the large subunit of human caspase- 14
(Santa Cruz Biotechnology). - C20 antibody directed to the small subunit of caspase-14
(Santa Cruz Biotechnology). - pQE 30 vector (Qiagen).
- pET15b vector.
- BL21 (DE3) bacterial strain (Novagen).
- Overnight Express Autoinduction System 1 (Novagen, Merck
KGaA). - Ni-NTA His Bind Resin (Novagen) or similar.
- Ni-NTA Agarose.
- Active KLK7 (e.g., from R & D systems).
- KLK7 assay buffer: 0.1 M HEPES or Tris–HCl (pH 7.5),
1 mM CaCl 2. - 0.1 M glycine-HCl buffer (pH 2.3).
- ImmunoBlock (DS Pharma Biomedical Co) or similar blocking
reagent. - PBS containing 0.1 % Tween 20 (PBST).
- ECL™ Anti-rabbit IgG Horseradish Peroxidase linked F(ab)′ 2
fragment (GE Healthcare) or similar. - TMB Peroxidase EIA Substrate Kit (Bio-Rad Laboratories).
- 0.1 M sulfuric acid.
- Acetone.
- Methyl benzoate.
- Xylene.
- Paraffi n (melting point 58–60 °C).
- Goat serum.
- VECTASHIELD Mounting Medium (VECTOR Laboratories).
- 4 ′,6′-Diamidino-2-phenylindole (DAPI).
Mami Yamamoto-Tanaka and Toshihiko Hibinohttp://www.ebook3000.com