82- Pellet cells by centrifugation at 200 × g for 5 min and wash in
cold PBS. - Resuspend cell pellet in cell extraction buffer (at approximately
1 × 10^7 cells/ml). - Snap freeze cells in liquid nitrogen and then thaw in ice-cold
water. Repeat this three times. - Protein extracts are cleared by centrifugation at 15,000 × g for
10 min at 4 °C and cleared supernatant (cytosolic extract) is
transferred to a clean tube. - Protein concentrations are determined using standard BCA
assays (Bio-Rad) and extracts are stored at −70 °C in small ali-
quots. Protein extracts and caspase activity are stable for several
months at −70 °C. - To observe caspase-2-mediated cleavage of cellular protein
substrates, incubate cell extract containing 20–30 μg total pro-
tein with 100 nM recombinant caspase-2 at 37 °C for 2–3 h in
a total volume of 50 μl of Caspase-2 Assay Buffer. - To stop the reaction, add 50 μl of 2× PLB, boil for 5 min, and
centrifuge at 10,000 × g for 5 min to remove any insoluble
material. - Resolve proteins on a 12–15 % polyacrylamide/SDS gel, and
transfer proteins to PVDF membrane using a wet or semidry
protein transfer apparatus. - Incubate PVDF membrane in 3 % skim milk (or 3 % BSA) in
PBST for 1 h at room temperature or overnight at 4 °C. - Dilute primary antibody as suggested by the manufacturer in
1–5 % skim milk/PBST (or 3 % BSA) and incubate the PVDF
membrane with the antibody solution for 1 h at room tem-
perature with gentle shaking. Incubation with caspase-2 anti-
body will show cleavage of caspase-2 and can be used as a
control to demonstrate activity of the recombinant caspase-2
protein ( see Note 3 ). Cellular proteins known to be cleaved by
caspase-2 are Bid, Golgin-130, and Mdm2 and can be detected
using antibodies specifi c to these proteins. - Wash the membrane twice for 5 min each and then twice for
10 min each and incubate with the appropriate secondary anti-
body diluted in 1–5 % skim milk/PBST (or 3 % BSA). - Protein detection can be carried out using ECL or ECF (as per
instruction supplied by manufacturer) and visualized on X-ray
fi lm (for ECL) or for ECF imaging on a fl uorescence scanner
bed (such as a Typhoon FLA 7000 Image Scanner, GE
Healthcare).
Affi nity labeling of caspase-2 can be carried out in cells in culture
following apoptotic stimulation or can be carried out using
apoptotic cell extracts. The affi nity label biotin-VAD-FMK is a3.4 Assessing Active
Caspase-2 In Vivo by
Affi nity Labeling
Loretta Dorstyn and Sharad Kumarhttp://www.ebook3000.com