24
- Using a pipettor, gently resuspend the cells and transfer them
to a 15 mL conical tube. - Recover the cells by centrifugation at 1,000 × g for 5 min at
4 °C. Discard the supernatant. - Wash the cells using 5 mL of cold PBS.
- Recover the cells by centrifugation at 1,000 × g for 5 min at
4 °C. Discard the supernatant. - Resuspend the cell pellet in 0.5 mL of eukaryotic lysis buffer,
transfer to a 1.5 mL microfuge tube, vortex for 10 s and incu-
bate on ice for 30 min (see Note 33). - Centrifuge the lysate at 7,000 × g for 10 min at 4 °C. Transfer
the supernatant to a new microfuge tube. - Determine the protein concentration using a protein assay that
is compatible with the eukaryotic lysis buffer (e.g., Pierce BCA
protein assay). - Dispense into 30 μL aliquots and freeze at −80 °C.
- Thaw an aliquot of active site titrated caspase and lysate (this
step takes approximately 30 min) on ice. Do not heat the
samples. - Set up 8 samples of 25 μL of a 2/3 serial dilution of the caspase
in 1× caspase buffer, starting with a caspase concentration of
100 nM. Include one sample with buffer only (sample 9).
Incubate for 5 min at 37 °C (see Note 34). - Prepare 0.25 mL of a 2 mg/mL lysate solution (or 200 nM of
purified protein substrate) in 1× caspase buffer and incubate at
37 °C for 5 min (see Note 35). - With a repeating pipettor, add 0.25 μL of diluted lysate to each
of the samples, mix, and incubate for 30 min at 37 °C. - Stop the reaction by adding 0.5 volume of 3× gel loading
buffer. - Analyze the samples by immunoblotting using an antibody
directed against the protein of interest. - Using imaging software, determine the concentration of cas-
pase at which ~50 % of the substrate is cleaved. Use this value
to determine k using Eq. 4 (see Note 36). - Repeat the experiment and adjust caspase concentration, lysate
concentration, time, or a combination thereof to set the ~50 %
cleavage sample between samples 4–6 (see Note 37).
Caspase-2
Caspase-2 has an extended substrate-binding pocket that recog-
nizes amino acids in position P5 (Schechter-Berger nomenclature
[ 24 , 25 ]). Consequently, AcVDVAD-Afc is used to characterize
the activity of this caspase. However, because of that extended3.3.2 Determining
the Kinetic Parameter
kcat,app/ KM,app for a Protein
Substrate
3.4 Particularities
of Caspases
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