241- 96-well fl at bottom tissue culture plates. Using black opaque
plates will decrease the crosstalk and background noise during
fl uorescence detection. - Plate reader to detect fl uorescence at excitation/emission 360–
380 nm/440–460 nm (Omega Fluostar, BMG or similar). - Materials described under items 1 – 5 in Subheading 2.2.
- Table centrifuge at 4 °C.
- Reaction buffer: 50 mM HEPES pH 7.5, 0.1 % (w/v) CHAPS,
5 mM DTT, 100 μM PMSF, 10 μM pepstatin A. - 1× Laemmli buffer: 50 mM Tris–HCl pH 6.8, 1 % (w/v)
sodium dodecylsulfate (SDS), 4 % (v/v) glycerol, 0.75 M
β-mercaptoethanol, 0.005 % bromophenol blue. Prepare a 5×
stock, store at room temperature. Add β-mercaptoethanol to
an aliquot right before use. - 12 % Resolving gel: 12 % Acrylamide–Bis-acrylamide (29:1),
375 mM Tris–HCl pH 8.8, 0.1 % (w/v) SDS. - 4 % Stacking gel: 4 % Acrylamide–Bis-acrylamide (29:1),
125 mM Tris–HCl pH 6.8, 0.1 % (w/v) SDS. - SDS-PAGE Running buffer: 25 mM Tris base, 24 mM gly-
cine, 0.1 % (w/v) SDS. - Transfer buffer: 25 mM Tris base, 24 mM glycine, 10 %
ethanol. - PBST: 4 mM Na 2 HPO 4 , 4 mM NaH 2 PO 4 , 1.5 mM KH 2 PO 4 ,
137 mM NaCl pH 7.2, 0.1 % (v/v) Tween-20. - Secondary antibody (Amersham, RPN 4201, RPN 4301, or
similar). - ECL (Amersham RPN2232 or similar).
- Coomassie Brilliant Blue Solution: 1/4.5/4.5 (v/v/v) glacial
acetic acid–ethanol–water, 0.05 mg/mL Coomassie Brilliant
Blue G250. - Stripping buffer: 25 mM glycine, 1 % (w/v) SDS.
- Equipment for SDS-PAGE and protein transfer.
- PVDF membrane (Bio-Rad 162-0177 or similar).
- Table centrifuge.
- TNT ® Coupled Transcription/Translation System (Promega)
( see Note 2 ). - Plasmid (0.5 μg/μl) containing the coding sequence (CDS)
for the metacaspase protein substrate of interest in a suitable
expression vector ( see Note 2 ).
2.3 Measurement
of Metacaspase
Activity in Cell Lysates
2.4 Detection
of Metacaspase
Activation In Vivo
2.5 Metacaspase
Protein Substrate
Cleavage Assay
Plant Metacaspases