Caspases,Paracaspases, and Metacaspases Methods and Protocols

250

1. Produce the protein substrate in the cell free TNT ® Coupled
   Transcription/Translation System (TNT ® mix) according the
   manufacturer’s instructions (Promega). A choice can be made
   here to use radiolabeled L -[^35 S]methionine for subsequent
   readout by autoradiography or unlabeled methionine and epi-
   tope tags for western blotting.


2. Prepare the metacaspase reaction master mix by mixing 80 μL
   of 4× reaction buffer with 120 μL of dH 2 O. Divide 25 μL per
   well over an 8-well eppi strip.


3. Add 5 μL of the TNT ® mix to the side of each well. Take care:
   the drop of TNT ® mix should not touch the metacaspase reac-
   tion mix.


4. The reaction is started by the simultaneous addition of 10 μL
   of the rAtMC4 dilution series to the 8-well eppi strip with the
   help of a multichannel pipette and by pipetting the metacas-
   pase reaction and TNT ® mix up and down. The reaction is
   carried out at 30 °C for 30 min.


5. The reaction is stopped by the simultaneous addition of 10 μL
   of the 5× Laemmli buffer + EGTA ( see Note 10 ) to each well.
   The samples are heated for 5 min at 85 °C and can be stored
   at −20 °C or immediately used in the subsequent steps.


6. Load 15 μL of each sample on a 12 % SDS-PAGE gel and run
   the electrophoresis.


7. After electrophoresis, rinse the gel shortly in dH 2 O and dry
   for 1 h on a Whatman paper in a gel dryer when using autora-
   diography as a readout, or proceed to western blot as detailed
   in Subheading 3.4 with the appropriate antibody against the
   used epitope tag.


8. For autoradiography, expose the dried gel for 48 h to a phos-
   phor screen and make the readout on a phosphor imager with
   appropriate settings ( see Note 11 ).

Fig. 3 In vitro metacaspase protein substrate cleavage assay. A potential substrate

protein is cleaved by an increasing concentration of recombinant Arabidopsis

thaliana metacaspase 4 (rAtMC4) to discrete cleavage products ( top panel ).

Glutathione S-transferase (GST) does not get cleaved by metacaspase ( bottom

panel ). Concentrations are indicated in the fi gure. Inactive rAtMC4, in which the

active site cysteine is mutated to alanine (C139A), acts as a negative control

Elena A. Minina et al.

http://www.ebook3000.com