Chemiluminescent Western Blot Assay for Protein Processing
Transcrição
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.
