85- There are also several commercial suppliers of purifi ed
recombinant caspase-2, such as Enzo Life Sciences (USA),
Millipore (USA), and MP Biomedicals (USA). - For caspase activity assays, if sample is highly active, the sub-
strate will be rapidly saturated. In this case, dilute out protein
or lysate to get a linear response. - There are several different methods describing affi nity capture
of active caspases, and the protocol described here is a modifi ca-
tion of the methods described in Faleiro et al. and in Dorstyn
et al. [ 38 , 40 ]. There are various caspase affi nity labels available,
and we have found that biotin-VAD-FMK labeling works best
to detect active caspase-2. Incubating cells with biotin-VAD-
FMK prior to cell lysis seems to be more effective in our hands. - If nonspecifi c binding is a problem, then this can be mini-
mized by including a wash step in lysis buffer containing 0.5 M
NaCl. However, this will need to be followed by additional
2–3 washes in lysis buffer (without NaCl) to reduce the high
salt concentration that may interfere with electrophoresis of
samples.
Acknowledgments
The fi nancial support of the National Health and Medical Research
Council through project grants 1021456 and 1043057 is gratefully
acknowledged. LD is supported by a South Australian Cancer
Research Collaborative Fellowship and SK by an NHMRC Senior
Principal Research Fellowship (1002863).References
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