20
monomers (inactive) from bacteria [ 27 – 30 ], the assay conditions
must somehow force the dimerization of the caspase. To re-create
this condition in vitro, a buffer that favors dimerization, such as
one that contains kosmotropic salts (e.g., sodium citrate), is used
[ 30 , 31 ]. In addition to promoting dimerization, kosmotropic
salts will also promote ordering/stabilization of crucial loops
implicated in caspase activity.
Practically, to titrate a caspase preparation, a series of enzy-
matic reactions are set up in a micro-well plate with a serial dilution
of Z-VAD-fmk. One reaction without the titrant is included. If the
highest final Z-VAD-fmk concentration is 1 μM, a 2/3 serial dilu-
tion over 15 wells will cover values between 3.4 and 1,000 nM of
caspase. For Z-VAD-fmk to inactivate as much caspase as possible
in a reasonable time, relatively high concentrations of enzyme are
required. These concentrations are necessary because, although
Z-VAD-fmk is a pan-caspase inhibitor, it is not potent for all cas-
pases. Most wild-type or truncated caspases will be inhibited within
30 min if 100 nM of enzyme is used in the appropriate assay buffer.
After the inhibition reaction, aliquots of reactions are transferred
to a new plate containing an appropriate substrate. The hydrolysis
of that substrate is measured continuously.
This procedure takes 2 h to be completed.
Protocol- Thaw an aliquot of caspase on ice (this step takes approximately
30 min). Do not heat the sample. - In a 96-well plate, set up a 2/3 serial dilution of Z-VAD-fmk in
1× caspase buffer (50 μL/well, starting at 2 μM) over 15 wells.
Set up one sample with buffer only (well 16) (see Note 24). - Based on the estimated caspase concentration, prepare 1 mL of
a 200-nM solution of caspase in 1× caspase buffer. Add 50 μL
of this solution to every well of the plate. The highest concen-
tration of Z-VAD-fmk will be 1 μM, and the final concentra-
tion of caspase will be 100 nM. - Thoroughly mix using a microplate mixer or the mixing func-
tion of the plate reader. Seal the plate with paraffin film and
incubate for 30 min at 37 °C. - Once the incubation is over, transfer an aliquot of the reaction
to a new series of wells containing 1× caspase buffer (final vol-
ume of 80 μL). Thoroughly mix and incubate the plate for
5 min at 37 °C (see Note 25). - During this time, prepare 350 μL of a 500 μM solution of cas-
pase substrate in 1× caspase buffer (see Note 26). - With a repeating pipettor, rapidly add 20 μL of the substrate
solution to each well. Thoroughly mix and immediately read the
fluorescence at EXλ = 405 nm and EMλ = 510 nm for 30 min at
37 °C. Ideally, take measurements every 5 s (see Note 27).
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