209All inhibitors for testing (e.g., Z-VRPR-FMK) should be made up
in 100 % DMSO or ddH 2 O at a concentration of 10–100 mM,
depending on individual inhibitor solubility.- Temperature controlled shaking incubator set at 37 °C.
- 1.8 mL microcentrifuge tubes.
- Working Buffer (for buffer exchange): 10 mM Tris pH 7.5,
50 mM NaCl, 5 mM DTT. - 10× Working Buffer: 100 mM Tris pH 7.5, 500 mM NaCl,
50 mM DTT. - 10 mM EGTA stock solution, prepared in ddH 2 O, pH-
adjusted to around 8.0 with NaOH and stored at room
temperature. - 10 mM CaCl 2 , prepared in ddH 2 O and stored at room
temperature. - Metacaspase peptide inhibitors, e.g., Z-VRPR-FMK (Enzo
Life Sciences) prepared to ~500 μM. - SDS-Sample Buffer (90 μL NuPAGE LDS Sample Buffer sup-
plemented with 10 μL NuPAGE Reducing Agent).
3 Methods
Sterile techniques and equipment should be used throughout cell
transformation and protein expression.- Thaw 50 μL of the chemically competent E. coli on ice.
- Add 5–10 ng of the expression plasmid DNA ( see Note 10 )
(typically 1–5 μL) to the cells and mix by tapping gently. - Place the cells on ice and incubate for 30 min.
- Transform the cells by heat-shock, by placing the vial at 42 °C
for 30 s. - Return the tube to ice for 2 min to allow the cells to recover.
- Add 250 μL of prewarmed SOC medium ( see Note 11 ) to the
vial and incubate at 37 °C with shaking (~225 rpm) for 1 h. - Pipette 20–200 μL ( see Note 12 ) onto the prepared and pre-
warmed LB Agar plates and spread the samples. - Invert the plates and incubate overnight at 37 °C.
- Aliquot 2 × 10 mL of the prepared auto induction medium
(containing kanamycin) into sterile tubes, to be used for starter
cultures, before dividing the remaining medium into four 1 L
baffl ed fl asks (~250 mL per fl ask). - Pick a single colony from an LB Agar plate and inoculate the
two starter cultures.
2.7 Inhibitor Studies
2.8 Auto-processing
and Inhibitor Binding
Gel-Shift Assays
3.1 Expression
3.1.1 Transformation
3.1.2 Metacaspase
Expression
Trypanosoma Metacaspases