This article discusses the role of CD95 in apoptosis through the formation of a death-inducing signaling complex (DISC). It details the process of analyzing caspase-8 processing using chemiluminescent western blotting.
CD95 ― a death receptor ― binds to its agonistic antibodies, triggering the assembly of a death-inducing signaling complex, or DISC ― a multiprotein complex that recruits procaspase 8. At the DISC, the procaspase 8 dimerizes and processes to form various intermediate cleavage products and, finally, an active form that initiates apoptosis.
To analyze caspase-eight processing using chemiluminescent western blotting, take a lysate containing DISC-associated intermediate cleavage products of caspase 8 in a tube. Supplement this with protein A-agarose bead-captured anti-CD95 antibodies. The anti-CD95 antibodies bind to the DISC component ― CD95, forming protein complexes.
Load the sample on an SDS-polyacrylamide gel to separate the different-sized immunoprecipitated caspase 8 intermediates as distinct bands. Place the gel on a blotting membrane, and apply an electric current. The caspase 8 intermediates move out of the gel onto the blotting membrane.
Treat the membrane with a blocking protein-containing solution to saturate the protein-binding sites ― preventing non-specific bindings.
Add a solution of primary antibodies, which bind to the caspase-eight products at a specific site.
Add enzyme-tagged secondary antibodies that specifically bind to the complementary Fc region of the primary antibody to form a caspase-eight cleavage product-primary antibody-secondary antibody complex.
Add a chemiluminescent substrate that reacts with the secondary antibody-conjugated enzyme and emits light. The intensity of the emitted light correlates with the caspase-eight cleavage product quantity.
To perform the western blot, add 20 microliters of 4X loading buffer to the beads, and heat at 95 degrees Celsius for 10 minutes. Simultaneously, heat the lysate controls at 95 degrees Celsius for 5 minutes. Load the lysates, immunoprecipitants, and a protein standard onto a 12.5% SDS gel, and run with a constant voltage of 80 volts.
After the gel run is complete, transfer the proteins from the SDS gel to a nitrocellulose membrane. After the transfer is complete, place the blotted membrane in a box, and incubate it for an hour in blocking solution, under gentle agitation. Then, wash the membrane three times for 5 minutes with PBST.
To detect the proteins, add the first primary antibody at the indicated dilution to the membrane, and incubate it overnight at 4 degrees Celsius with gentle agitation. The next day, wash the membrane with three 5-minute PBST washes, then, incubate the membrane with 20 milliliters of secondary antibody with gentle shaking for 1 hour at room temperature.
Wash the membrane three times with PBST again. After discarding the last PBST wash, add approximately 1 milliliter of horseradish peroxide substrate to the membrane, and detect the chemoluminescent signal.
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