76
- Centrifuge.
- PBS.
- Chromatography columns (such as Bio-Rad Poly-prep chro-
matography columns). - Ice.
- Sonicator.
- Glutathione Sepharose for the purifi cation of GST-tagged pro-
teins or Ni-NTA beads for the purifi cation of 6×His-tagged
proteins. - Elution Buffer: GST—50 mM Tris–HCl pH 8.0, 10 mM
reduced glutathione; Ni-NTA—50 mM sodium phosphate pH
7.0, 300 mM NaCl, 200 mM imidazole. - Caspase-2 synthetic peptide substrate: peptide substrates com-
prising an N -acetyl (Ac)- and C-terminal-conjugated 7-amino- 4-
trifl uoromethylcoumarin (AFC) or 7-amino-4-methylcoumarin
(AMC) reporters. Ac-Val-Asp-Val-Ala- Asp-AMC/AFC (Ac-
VDVAD-AMC/AFC) is commonly used (available from vari-
ous commercial sources such as Calbiochem). Colorimetric
substrates with p -nitroanilide (pNA) reporter are also available
(but not as sensitive). AMC and AFC- conjugated substrates
are made up as 5–10 mM stock solutions in dimethyl sulfoxide
(DMSO) and can be stored in aliquots at −20 °C. - Caspase-2 peptide inhibitors: commonly conjugated to chloro-
methylketone (CMK), fl uoromethyl ketone (FMK), or CHO-
aldehyde and act as irreversible inhibitors with no added
cytotoxic effects. Commonly used (semi-specifi c) caspase-2
inhibitors include z-VDVAD-FMK and Ac-VDVAD-CHO.
Caspase inhibitors are made up at 5–10 mM stock in DMSO
and stored in aliquots at −20 °C. Other commonly used pan-
caspase inhibitors are z-valine-alanine-aspartate-fl uoromethyl
ketone (z-VAD-FMK) and quinolyl-valyl- O -methylaspartyl-
[-2,6-difl uorophenoxy]-methyl ketone (Q-VD-OPh). Avoid
repeated freeze–thaw cycles. - Caspase-2 Assay Buffer: 0.1 M (2-[ N -morpholino]ethanesul-
fonic acid) (MES), pH 6.5, 10 % sucrose, 0.1 % CHAPS,
0.5 mM EDTA, supplemented with 10 mM DTT and protease
inhibitors ( see Notes 1 and 2 ). - Liquid nitrogen.
- Ice.
- Bench top centrifuge.
- 96-well plate.
8. Fluorometric plate reader equipped with a thermostated plate
reader, such as a FLUOstar Optima Luminescence Spectrometer
(BMG LabTech).
2.4 Fluorogenic
Caspase-2 Activity
Assays
Loretta Dorstyn and Sharad Kumarhttp://www.ebook3000.com