78
- Power pack supply with constant current optional settings.
- Wash buffer: PBS with 0.05 % Tween-20 (PBS-T).
- Membrane blocking buffer: 3 % nonfat milk or 3 % bovine
serum albumin (BSA) in PBS. - Caspase-2 antibody: 1 μg/ml anti-caspase-2 rat monoclonal
antibody (clone 11B4) diluted in 3 % skim milk in PBS-T
( see Note 3 ). - Secondary antibody: anti-rat secondary antibody conjugated
to alkaline phosphatase (AP) or horse radish peroxidase (HRP). - Protein detection solutions: enhanced chemifl uorescence
(ECF) (AttoPhos, GE Healthcare/Amersham) or enhanced
chemiluminescence (ECL) (ECL Plus GE Healthcare/
Amersham). - X-ray fi lm and an X-ray developing machine for ECL
detection. - Laser scanner for fl uorescent detection of proteins, such as the
Typhoon FLA 7000 Image Scanner for ECF detection (GE
Healthcare/Amersham). - Affi nity label biotin-VAD-FMK is prepared as 10 mM stock in
DMSO, aliquoted and stored at −70 °C. - Affi nity Label Dilution Buffer: 50 mM HEPES-KOH pH 7.0,
50 mM NaCl, 1 mM MgCl 2 , 1 mM ethylene glycol-bis
[β-aminoethyl ether]- N , N , N ′, N ′-tetraacetic acid (EGTA),
1 mM EDTA, 1 mM DTT, and supplemented with protease
inhibitor cocktail. - Cell Lysis Buffer: 50 mM HEPES-KOH pH 7.0, 50 mM
NaCl, 1 mM EDTA, 1 mM MgCl 2 , 5 mM DTT, 0.1 % CHAPS,
1 mM phenylmethanesulfonyl fl uoride (PMSF), and supple-
mented with protease inhibitor cocktail. - Streptavidin Sepharose.
- 2× Protein Loading Buffer (PLB).
- Rotating platform at 4 °C.
- Bench top centrifuge.
3 Methods
- A bacterial expression plasmid containing the caspase-2 coding
region in frame of a C-terminal 6×His tag (pET-Caspase-2) or
GST-tag (pGEX-Caspase-2) ( see Note 4 ) is transformed into
BL21 cells (typically cells that express lysozyme such as BL21-
star or BL21-pLysE) using a standard heat shock method, and
2.8 Affi nity Capture
of Active Caspase-2
3.1 Purifi cation of
Active Recombinant
Caspase-2
Loretta Dorstyn and Sharad Kumarhttp://www.ebook3000.com