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Related Links Caspase Cleavage and Activation Physiological Caspase Substrates and Inhibitors Mitochondrial Changes in Apoptosis |
Antibodies are classic tools used to visualize caspase processing by Western blotting. Applications such as flow cytometry and microscopy require antibodies that specifically recognize either the pro- or cleaved caspase forms because these techniques are unable to resolve multiple protein forms with a single antibody (see figure below). The bands observed on a Western blot to visualize caspase processing will depend on the epitope recognized by a given antibody and the caspase forms present.
Detection of Caspase Processing A) To induce apoptosis, Jurkat cells were treated with 2 uM Staurosporine (STS) for the indicated times, and Caspase-3 processing was monitored by Western blots. The pro-caspase (full length, ~32 kDa) and the large subunit of processed human Caspase- 3 (~17 kDa) were detected using Caspase-3 monoclonal antibody (catalog # NB100-56708). A fragment of cleaved PARP (~89 kDa) was detected using PARP monoclonal antibody (catalog # NB100-56599). Note that Caspase-3 and PARP were processed only in cells treated with STS. B) Jurkat cells were treated as in A, but Caspase-3 processing was visualized by multicolor immunocytochemistry /immunofluorescence (ICC/IF) using cleaved Caspase-3 monoclonal antibody (catalog # MAB7071) followed by NorthernLights™ 557-conjugated secondary antibody (catalog # NL004), shown in red. Nuclei are stained with DAPI, shown in blue. Guidelines for Monitoring Caspase ProcessingIf the loss of pro-forms, e.g. of Caspase-3, is not accompanied by or is proportional to cleaved Caspase-3, the loss itself is considered an indicator of caspase processing. It is important to note that bands representing cleaved caspase fragments may be transient due to their short half-life and are not definitive markers of caspase activation. Confirmation of caspase activation can include immunodetection of processed physiological substrates such as PARP-1. |
