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Metacaspase activity in vivo can be also assessed by analyzing
the effi ciency of natural substrate cleavage, e.g., cleavage of TSN by
mcII-Pa [ 18 ]. TSN is a highly conserved protein and anti- human
TSN antibodies are commercially available, but their interaction
with TSN from other species should be evaluated in the fi rst place.
We have optimized detection of mcII-Pa activation in vivo
during developmental and oxidative stress-induced cell death using
custom produced antibodies (Fig. 2 ).- Grind plant material in liquid nitrogen.
- Mix 100 mg of the ground material with 100 μL of the
reaction buffer without calcium and vortex. - Add Laemmli buffer and boil the samples for 10 min at
100 °C. - Centrifuge the samples at 17,000 × g for 10 min.
- Transfer the supernatant into new Eppendorf tubes.
- Depending on the tissue, extraction furnishes 1–5 mg/mL of
total protein. Load 5–15 μL of each sample on 12 % polyacryl-
amide gel and run electrophoresis. - Transfer proteins on PVDF membrane.
- Block the membrane for 10 min in 5 % skim milk.
Fig. 2 Autoprocessing of Norway spruce metacaspase mcII-Pa and cleavage of TSN detected by immunoblot-
ting. ( a ) mcII-Pa was activated by transferring embryogenic cells from the medium containing plant growth
regulators (PGR; +) to the medium devoid of PGR (−). Zymogen and auto-cleavage products of mcII-Pa were
detected by using custom produced mcII-Pa-specifi c antibody, as described in the protocol. Pattern of the
mcII-Pa autoprocessing has been assessed by N-terminal sequencing of products of cleavage [ 15 ] ( b ) mcII-Pa
was activated by inducing oxidative stress in the 4-day-old cell culture using 10 mM H 2 O 2 ; the samples were
taken at 0, 10, 16, and 24 h of treatment. Activity of mcII-Pa was assessed by detecting products of TSN cleav-
age using custom produced TSN-specifi c antibody as described in the protocol
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