Mammalian caspases can be subdivided into three functional groups: initiator caspases (caspase 2, 8, 9 and 10), executioner caspases (caspase 3, 6 and 7), and inflammatory caspases (caspase 1, 4, 5, 11 and 12). Initiator caspases initiate the apoptosis signal while the executioner caspases carry out the mass proteolysis that leads to apoptosis. Inflammatory caspases do not function in apoptosis but are rather involved in inflammatory cytokine signaling and other types of cell death such as pyroptosis.
Initially synthesized as inactive pro-caspases, caspases become rapidly cleaved and activated in response to granzyme B, death receptors and apoptosome stimuli. Caspases will then cleave a range of substrates, including downstream caspases, nuclear proteins, plasma membrane proteins and mitochondrial proteins, ultimately leading to cell death.
Activation of caspases can be detected using multiple methods, and the answer provided by different experimental methods will confirm whether one or more specific caspases are active or inactive. It is always best practice to use more than one methods to confirm specific caspase activation. Here we show how to detect activation of caspase 3 using a combination of antibody and substrate-based methods.
The ability to detect active caspase relies on the specificity of the antibody and where the epitope is located. Therefore, it is key to choose the right antibody for the right method.
Figure 1. Detection of caspase-3 by western blot. HeLa cells were left untreated (lanes 1, 3, 5, 7) or treated (lanes 2, 4, 6, 8) with 1 μM staurosporine for 4 hours. Apoptotic proteins (PARP and caspase 3) and a loading control (actin) were detected with Apoptosis Western Blot Cocktail (ab136812).
The antibody against caspase 3 used in figure 1 recognizes an epitope found in both uncleaved (proenzyme) and cleaved (active form) caspase 3. Western blot analysis is the recommended assay for such antibodies as it allows differentiation of both form and also allows bands to be used as transfection controls (ie there has been no issue in the transfer process).
To determine caspase 3 activation by immunostaining (figure 2), it is more appropriate to use an antibody that only detects the cleaved active form so that only apoptotic cells are stained.
Figure 2. Detection of active caspase 3 by immunofluorescence. HeLa cells were left untreated (left) or treated with 1 µM staurosporine for 4 hours (right) and stained with Active Caspase 3 antibody [E83-77] (ab32042) at 1:100 dilution.
Biochemical substrates consist of short peptides that contain a specific cleavage sequences that are recognized by the caspase and are covalently attached to a colorimetric or fluorogenic detection probe. Upon cleavage of the substrates by the cognate caspase, the colorimetric or fluorogenic compound is liberated producing an increase in absorbance (colorimetric substrate) or fluorescence light (fluorogenic substrate). The resulting signal is proportional to the amount of caspase activity present in the sample; however, many of the cleavage sites are similar and can be cleaved by other caspases. For example, caspase 3 and 7 have very similar cleavage sites. In these situations, the use of specific activators and/ or inhibitors of caspase activity is essential to ensure determination of the correct activity.
Another important consideration when looking at caspase activation is to ensure that the specific caspase is activated in the sample you have chosen. For example, the breast cancer cell line MCF-7 lacks a functional caspase 3, therefore, determination of caspase 7 activity would be preferable.
Figure 3. Caspase 3 activity in Jurkat cells. Jurkat cells (3.3 x 106 cells) were lyzed following 20 hour exposure to 2 µM camptothecin (CPT) or 10 ng/mL anti-Fas antibody (a-Fas). Untreated cells were used as control (CTRL). Caspase 3 activity was detected using Caspase 3 Activity Assay Kit (Colorimetric) (ab39401).
This table outlines the most common methods used to detect caspase activity:
|Sample type||Detection method||Best to use when you want to ....||Products|
|Live cells (suspension or adherent)||Flow cytometry||Quickly detect and quantify how many cells have active caspase using a specific substrate||ab112130|
Fixed cells (suspension or adherent)
|Fluorescence microscopy||Visualize which cells have active caspase. Commonly used when you want to visualize other proteins at the same time||ab65613|
|Flow cytometry||Quickly detect and quantify how many cells have active caspase using a specific antibody||ab65613|
Cell or tissue lysates (fresh or frozen)
|Western blot||Detect a caspase in its cleaved form as well as in its pro-caspase form using specific antibodies|
|Absorbance / fluorescence assay||Quickly detect caspase activation in a cell population using a specific substrate. Easily adaptable for HTP analysis.|
|Absorbance / fluorescence assay||Quickly detect caspase activation in a cell population using a specific antibody against the active form.|
|Tissue section||IHC||Visualize caspase|
activation with a specific antibody in discrete cells in a heterogeneous tissue (patient sample, mouse or rat tissue).
Learn more about apoptosis at www.abcam.com/apoptosis