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recombinant caspases. We have found that the rat anti-caspase-2
antibody clone (11B4) works best to detect full-length cas-
pase- 2 and cleaved 37 kDa caspase-2 (minus pro-domain)
(Fig. 3 ). Some antibodies, such as rabbit anti-caspase-2 (C10,
Santa Cruz Biotechnology, Santa Cruz, CA, USA) will detect
both the precursor and one or more cleavage products (Fig. 3 ),
while others only appear to recognize full-length caspase-2.
Active caspase-2 antibody is available from some companies
(Cell Signaling) and will detect cleaved small subunits (p19
and p14/p12). In some cell types, the half-life of processed
caspase- 2 subunits can be short, but a decrease in zymogen
signal can generally be monitored to determine whether cas-
pase-2 is cleaved following specifi c stimuli.- Expression of a caspase-2 protein without the pro-domain
(i.e., minus CARD) in bacteria has been demonstrated to have
higher activity than full-length caspase-2 protein. - GST or 6×His tags should be placed C-terminal to caspase-2
protein, since caspase-2 is rapidly processed following the
CARD, when expressed in bacteria. We have not found that
the GST or 6×His tag interferes with the activity of caspase-2;
however, it is also possible to cleave these tags provided the tag
is preceded by a thrombin cleavage site or TEV cleavage site.
Fig. 3 Processing of caspase-2 in apoptotic cell lysates. Jurkat cells ( a ) or mouse embryonic fi broblasts
(MEF) ( b ) were treated with etoposide (40 μM) and cell lysates prepared at the indicated time points over 24 h.
Protein was electrophoresed through 15 % ( a ) or 4–15 % SDS-PAGE gradient gel ( b ) and transferred to PVDF
membrane. Immunoblotting was carried out using anti-caspase-2 rabbit polyclonal antibody (SC-625; Santa
Cruz) to detect human caspase-2 ( a ) or with anti-caspase-2 rat monoclonal antibody (11B4, Millipore) to
detect mouse caspase-2 ( b ). β-Actin monoclonal antibody (Sigma) was used as a protein load control. Signals
were detected by ECL
Loretta Dorstyn and Sharad Kumarhttp://www.ebook3000.com