247- Dilute 5-μL aliquot of the supernatant ten times with Milli-Q
or dH 2 O water and use it for estimating total protein concen-
tration using Bradford kit. - Make two dilutions of the supernatant with reaction buffer
containing 50 mM CaCl 2 and without calcium. Final protein
concentration in the diluted samples should be 158 μg/mL. - Dilute AMC-conjugated substrate with reaction buffer to the
fi nal concentration 1 mM. - Prepare a blank sample by mixing 152 μL of reaction buffer
with 8 μL of 1 mM substrate. - Prepare fi ve standard samples by making serial dilutions of
24 μM AMC stock using reaction buffer to obtain 12, 6, 3,
1.5, and 0.75 μM of AMC. - Pipet 50 μL of each standard solution and the blank solution
into 96-well opaque plate (1,200, 600, 300, 150, 75, and
0 pmol of AMC per well), in triplicates. - Pipet 47.5 μL of diluted samples (from step 6 ) into 96-well
plate (7.5 μg of total protein per reaction), in triplicates. - Add 2.5 μL of 1 mM substrate to each well containing cell
lysate samples (fi nal concentration of substrate 50 μM). - Set the detector gain to 70 % using the 3-μM AMC standard.
- Set the temperature in the plate reader in the range 25–28 °C.
- Shake the plate in the plate reader for 10 s and take reads of
fl uorescence intensity at excitation/emission 360–
380 nm/440–460 nm every min making at least 20 fl ashes per
well. Take reads for at least 15 min. - Use the standard curve and linear part of the reaction curve to
calculate pmol of AMC released per minute per microgram of
total protein. - Subtract values obtained for samples without calcium from the
values of samples containing calcium, or present the values
without calcium as a control (Fig. 1 ).
Most metacaspases require autoprocessing for activation, thus
detection of metacaspase auto-cleavage products can be used for
assessing its activation in vivo. If metacaspase-specifi c antibodies
are available, autoprocessing can be directly estimated by western
blot analysis. Otherwise expression of a tagged metacaspase is
required for the assay. Detecting tagged metacaspase instead of
using metacaspase-specifi c antibodies is also preferable if there are
multiple metacaspases present in the sample to avoid crosstalk in
immunoblotting results.3.6 Detection
of Metacaspase
Activation In Vivo
Plant Metacaspases