7.1 Caspase 3

Caspase 3 is generally regarded as the primary terminal cleavage enzyme in the process of apoptosis, a key executor of apoptosis, and also an important component of the cytotoxic T lymphocyte (CTL) cell killing mechanism. This is because it exerts a cleavage effect on many crucial proteins, such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP). The activation of Caspase 3 requires a proteolytic process in which its inactive proenzyme form is converted into activated p17 and p12 fragments.

The most common issue with cleaved-Caspase 3 is the absence of bands during Western Blot (WB) detection. As we mentioned earlier, apoptosis or other stimuli are key factors inducing the cleavage of Caspase 3. Therefore, the first step is to ensure that the samples receive appropriate stimulus treatment. If cells undergo apoptosis, detection can be performed from aspects such as morphology, cell function, and biochemical indicators. If the cleaved form of Caspase 3 cannot be detected, the following methods can be used to further verify the apoptotic level of the samples. If no apoptosis is detected by other experimental methods either, we may need to consider whether the sample modeling and stimulation methods are appropriate.

If other experimental methods can clearly confirm the apoptosis of the samples, we need to troubleshoot the causes during the WB process:

Apoptosis Detection Category Detection Methods
Morphology Electron microscopy, PI staining, Hoechst staining
Cell Function Annexin V-PI staining, Cytochrome C (Cyto C) release, mitochondrial membrane potential
Biochemical Indicators Caspase 3, PARP, DNA ladder
Caspase 3 Troubleshooting Steps
1. Cleaved-Caspase 3 may translocate into the nucleus
Cleaved-Caspase 3 may translocate into the nucleus. During sample preparation, it is necessary to use a strong lysis buffer combined with sonication to increase the solubility of the target protein.
2. Whether the sample loading amount is sufficient
Check if the sample loading amount is sufficient; it is recommended to load more than 20 μg of protein.
3. Cleaved-Caspase 3 has a small molecular weight
Cleaved-Caspase 3 has a small molecular weight. Pay attention to the membrane transfer time and avoid over-transfer (i.e., prevent the protein from transferring through the membrane).
4. Whether the primary antibody can detect the cleaved form
Verify if the primary antibody is capable of detecting the cleaved form (pay attention to the antibody’s immunogen and specificity), and check if the primary antibody concentration and incubation time can be optimized.
5. Whether the ECL reagent is sufficiently sensitive
Check if the enhanced chemiluminescence (ECL) reagent has sufficient sensitivity.
6. Use a positive control with known apoptosis
Use a sample with clearly confirmed apoptosis as a positive control.