217- Add 100 μL ddH 2 O to 11 individual wells on single row (wells
1–11) of the 96-well plate. - In well 12 add 150 μL of the stock inhibitor.
- Using a pipette, remove 75 μL of the solution in well 12 and
place into the adjacent well (well 11) and mix by gently
pipetting. - Repeat this 2-fold dilution (transferring 75 μL from well 11 to
well 10 etc.) for the remaining wells. - Discard 75 μL of the diluted inhibitor from well 1.
- To the inhibitor solutions add the following reagents in order:
(a) 100 μL 2× Assay Buffer.
(b) 5 μL protein (40× working solution).
(c) 20 μL substrate. - Remove the plate from the ice, place in the fl uorescence plate
reader, and equilibrate to ambient temperature (20–25 °C)
for 5 min. - Read the fl uorescence using an excitation wavelength of
355 nm and emission wavelength of 460 nm, using time scales
and intervals described above. - Plot FU versus T for each concentration of inhibitor.
- Calculate the reaction rate for each inhibitor concentration
following the method set out in Subheading 3.5.1. - Use Grafi t or Prism to plot [inhibitor] versus reaction rate and
calculate the IC 50.
Many metacaspases exhibit in vitro auto-processing. This is typi-
cally enhanced in the presence of 1 mM CaCl 2 , inhibited by 1 mM
EGTA and can be visualized using SDS-PAGE.- Exchange the protein into the Working Buffer and concen-
trate to around 10 μM. - For each sample for analysis add:
(a) 10 μL of protein.
(b) 2 μL of EGTA or CaCl 2 (optional).
(c) 2 μL 10× assay buffer.
(d) ddH 2 O to 20 μL. - Incubate the samples for around 30 min at 37 °C with or with-
out shaking ( see Note 20 ). - Stop the reaction by adding 11 μL SDS-Sample buffer.
- Heat samples for 10 min at 70 °C or for 3 min at 90 °C.
- Load each sample onto a 4–12 % SDS-PAGE gel and run for
40 min in MES buffer.
3.5.6 In Vitro Auto-
processing of Active
Metacaspases and Mutant
Variants
Trypanosoma Metacaspases