249- Rinse the membrane in PBST buffer and incubate with primary
antibody for 1 h at room temperature or overnight at 4 °C. - Wash the membrane three times for 5 min in PBST.
- Incubate with secondary antibody (HRP-conjugated antibody
is preferable to get a strong signal). - Wash the membrane three times for 10 min in PBST.
- Detect the signal using ECL substrate.
- If the membrane was used for detecting metacaspase auto-
cleavage products it can be reused to detect cleavage of TSN
and vice versa ( see Note 7 ). Strip the antibodies from the
membrane by shaking it in stripping buffer at 37 °C for
15 min. Change the buffer twice. - Block the membrane in skim milk as at step 8 and proceed
with staining as usual. - For loading control, a housekeeping protein can be detected
by western blot analysis using the same samples or the mem-
brane can be stained with Coomassie. After developing the
chemiluminescent signal rinse the membrane in PBST and
incubate in Coomassie Brilliant Blue solution for 10–15 min. - Rinse the membrane in 70 % ethanol to get rid of excessive
staining ( see Note 8 ). - Let the membrane dry at room temperature.
- Scan the membrane. If required loading can be quantifi ed
using ImageJ or any similar software ( see Note 9 ).
Metacaspases, as all proteases, exert their function through the
cleavage of protein substrates. The following assay is a rapid and
easy way to prove the cleavage of potential metacaspase protein
substrates by recombinant metacaspase in vitro. Due to the mini-
mal requirements of input material (basically, a suitable plasmid for
cell-free protein expression containing the CDS for the protein
substrate of interest), a relative high-throughput can be achieved.
This is particularly interesting to screen many potential substrate
proteins coming from proteome-wide degradome studies, such as
from COFRADIC technology [ 17 , 21 ], or to screen point-mutated
variants of a single substrate protein. The readout is based on auto-
radiography of radiolabeled L -[^35 S]methionine containing pro-
teins, so best practices for work with radioactive isotopes should be
upheld. However, the readout can be replaced by western blotting
when using appropriate epitope tags. Recombinant Arabidopsis
thaliana metacaspase 4 (rAtMC4) is used as an example here
(Fig. 3 ), but the assay should be widely applicable to other meta-
caspases with similar reaction conditions. Because of the rAtMC4
concentration range the effi cacy of cleavage can be assessed between
protein substrates in a relatively quantitative manner.3.7 Metacaspase
Protein Substrate
Cleavage Assay
Plant Metacaspases