13- Analyze a 10 μL aliquot from each fraction by SDS-PAGE. Pool
the purest and most concentrated fractions. Proteins can be kept
3–4 days at room temperature before proceeding to refolding.
Optional: If higher protein concentration and purity are
required, the following DEAE ion exchange purification proce-
dure (steps 12– 16 ) is recommended. This procedure will help
to remove some cleaved caspase-8. - Pour 2 mL of DEAE Sephadex resin into an empty chroma-
tography column (1.0 cm or less in diameter; e.g., Bio-Rad
Econo-Pac 0.7 × 5.0-cm) and let the liquid drain by gravity
flow. Rinse the resin twice with 5 bed volumes of Milli-Q water
and then 5 bed volumes of urea buffer (DAY 6). - Apply the pooled fractions containing caspase-8 (step 11) to
the column and let drain. - Wash the column with 5 bed volumes of urea buffer and let
drain between each wash. - Elute the protein with a step gradient of NaCl in urea buffer
(0–400 mM; 10 fractions; 2 mL/fraction). - Analyze a 10 μL aliquot from each fraction by SDS-PAGE.
Pool the purest and most concentrated fractions. Proteins can
be kept 3–4 days at room temperature before proceeding to
refolding.
Note: Every step of this procedure is performed at room temperature.- Centrifuge the pooled fractions from step 11 or step 16 in
Subheading 3.1.2.2 for 15 min at 18,000 × g to remove insolu-
ble proteins and residual resin. Transfer the supernatant to a
dialysis tube (10,000 MWCO; prepared according to manufac-
turer instructions) equilibrated in refolding buffer #1 (DAY 6). - Dialyze overnight at room temperature against 1 L of refold-
ing buffer #1. Stir buffer at low speed. - The next day, dialyze for 5 h at room temperature against 1 L
of refolding buffer #2. Stir buffer at low speed (DAY 7). - Recover the dialyzed protein with a pipet.
- Centrifuge the protein solution for 30 min at 18,000 × g to
remove any insoluble protein. Transfer the supernatant to a
fresh tube. - Determine the final protein concentration using a protein assay
that is compatible with refolding buffer #2 (e.g., Pierce BCA
protein assay). Use fresh refolding buffer #2 as a blank.
Optional: Concentrate the caspase preparation using Millipore spin
concentrators (10,000 MWCO). - Active site titrate the caspase-8 according to Subheading 3.2.2.
Assume a refolding yield of 5–10 % (see Note 17).
Refolding Full-Length
Caspase-8
Apoptotic Caspases Assays