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- LED-based systems are more stable and should be calibrated
every 6 months, whereas lamp-based instruments should be
calibrated every other month. Recalibrate every time a perfor-
mance variation is suspected. - After this period, all available Afc substrate should be cleaved.
Although caspase-3 may not be the best enzyme to hydrolyze
a specific substrate, it is the most reliable caspase available, and
excess enzyme will compensate for a less optimal substrate.
Other caspases can be substituted, but the reaction must be
carefully monitored to ensure complete substrate hydrolysis
(monitored in step 3). - It is important that the reaction is complete when the absor-
bance is measured. Therefore, if the absorbance is not stable
after 1 h, it is recommended to incubate the reaction for an
additional 30 min and read the absorbance again. This proce-
dure works because the executioner caspase buffer does not
significantly affect the absorbance of Afc. If the buffer is modi-
fied, one must calibrate the substrate by comparing the 10 μM
hydrolyzed substrate sample (step 2) to dilutions of Afc standard
(Subheading 3.2.1.1) in the same buffer. - Several techniques can be used to generate a serial dilution of
a reagent. However, although it may not be the most accurate
way of doing so, the following approach works fine. Fifty
microliters of 1× caspase buffer is added to wells #2–16, 150 μL
of 2 μM Z-VAD-fmk is added to well #1, and 100 μL is trans-
ferred from well to well (wells #1–15) by repeatedly pipetting
up and down to mix. Use either executioner or initiator cas-
pase buffer throughout this procedure. - The final concentration of caspase that is used to determine the
uninhibited enzyme varies based on the intrinsic activity of the
enzyme: 1–2 nM caspase-3; 2–5 nM caspase-2, caspase-6, and
caspase-7; and 10–20 nM caspase-8, caspase-9, and caspase-10.
It is important to note that mutant caspases may display enzy-
matic activity different from that of the wild- type enzyme.
Thus, the volume of enzyme transferred must be tailored based
on the caspase. - We used a different substrate for each caspase (Table 2 ).
- Continuous reading is preferred to end point kinetics.
Continuous reading allows the accumulation of several data
points at early times in the assay that will provide a better esti-
mation of the initial rate. Continuous reading also allows for
the identification of potential substrate depletion, initial issues
with temperature reaction equilibration, and unusual behavior
such as sample precipitation. - Do not include values that show >90 % inhibition for the cal-
culation of the titer, as these values may cause an overestima-
tion of caspase concentration. It is important that the data
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