Journal: Cell Death & Disease
Article Title: Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach
Figure Lengend Snippet: Purified antibodies specifically recognize cleaved caspase substrates with a pattern of ‘DXXD'. Apoptosis was induced by 5-FU and TRAIL in HCT116 cells as described in ‘Materials and Methods'. QVD-OPH was added to block caspase activities as indicated. Cell lysate from 10 7 cells was immuno-precipitated with 20 μ g of purified antibodies, then detected with antibodies against PARP ( a ), caspase 6 ( b ) or cleaved CK-18 ( c ), separately. The purified antibodies specifically recognize not only the cleaved PARP (DEVD) and caspase 6 (DVVD) whose caspase cleavage sites are one of the eight peptides used for immunization, but also the cleaved CK-18 (DALD), whose tetrapeptide cleavage site was not one of the eight peptides, but has aspartic acid at the P1 and P4 position. However, the purified antibodies do not recognize the cleaved caspase 7 (IQAD) or lamin A (VEID) whose tetrapeptide cleavage sites has a ‘D' at P1 but not P4 position (data not shown). Rabbit IgGs in the immune precipitate were detected as artifacts in western blots (as labeled) by the secondary antirabbit antibodies. This was due to the fact that both the NEAs and the detection antibodies (i.e. anti-PARP and anti-caspase 6) were raised in the same species (rabbit). Furthermore, to avoid precipitation, samples were not boiled before loading onto gels, therefore IgG oligmers were seen on the blots. ( d ) Purified antibodies specifically pull down cleaved recombinant CK-18. A total of 10 μ g of recombinant CK-18 was cleaved with caspase 3 in vitro . QVD-OPH was added to block caspase activities as indicated. The reaction was precipitated with purified antibodies and then detected with antibody against cleaved CK-18. ( e ) Peptides with a ‘D' at P1, or P1 and P4 (IETD, LQTD, SECD, DCRD, FRHD and FAED) were coated on the ELISA plates, and probed with purified antibodies. Scramble 3-DETD and Scramble 3 peptides were coated as positive and negative control, respectively. The antibodies only recognize the peptides with aspartic acids at both the P1 and P4 position (i.e. DCRD and DETD), but not the ones with a ‘D' only at P1 position
Article Snippet: The following primary antibodies were used in immunoblotting: anti-PARP (Cell Signaling Technologies, Danvers, MA, USA), anti-Caspase 6 (Cell Signaling Technologies), M30 (Peviva through Diapharma, West Chester, OH, USA).
Techniques: Purification, Blocking Assay, Western Blot, Labeling, Recombinant, In Vitro, Enzyme-linked Immunosorbent Assay, Negative Control