Caspase-3 (CASP3)

Caspase-3 target protein structure.

Figure 1: Caspase-3 target protein structure.

Caspase-3 target introduction

Protein function

Caspase-3 is a cysteine-aspartic protease that plays a key role in the process of apoptosis and is widely used as a biomarker for apoptosis.
During apoptosis, Caspase-3 is cleaved into two fragments with molecular weights of 17 kDa (p17) and 12 kDa (p12) and translocated to the cell nucleus.
After cleavage and activation, Caspase-3 can activate caspase-6, -7, and -9, leading to a cascade of apoptosis.
Caspase-3 is involved in the cleavage of huntingtin.
Caspase-3 is involved in the cleavage of amyloid-like proteins and is associated with neuronal death in Alzheimer's disease.

Protein expression

Highly expressed in lungs, spleen, heart, liver, and kidneys.
Moderate levels in brain and skeletal muscles, low in the testis.
Also found in many cell lines, with highest expression in cells of the immune system.

Protein localization

Expressed in the cytoplasm and translocated to the cell nucleus upon stimulation.

Caspase-3 Immunocytochemistry/ Immunofluorescence - Anti-Caspase-3 antibody [E87] (ab32351) Green: Caspase-3, Red: Tubulin

Image 2: Caspase-3 Immunocytochemistry/ Immunofluorescence - Anti-Caspase-3 antibody [E87] (ab32351) Green: Caspase-3, Red: Tubulin.

Isoforms & post-translation modifications

Human (P42574): 32kDa (predicted)
Mouse (P70677): 31kDa (predicted)
Rat (P55213): 31kDa (predicted)
Cleavage by granzyme B, caspase-6, caspase-8 and caspase-10 generates the two active subunits.
Active heterodimers between the small subunit of caspase-7 protease and the large subunit of caspase-3 also occur and vice versa.
Inactivated caspase-3 is nitrosylated at its catalytic site and becomes denitrosylated upon the activation of the apoptotic pathway.

WB experiment tips

Precautions

Please note that there are multiple forms of caspase-3 protein, and the degree of apoptosis induced in different samples may vary, resulting in differences in the size of the detected protein bands, including the approximately 32 kDa caspase-3 precursor (some samples may observe intermediate cleavage products of 29 kDa), the approximately 17 kDa cleaved form (some samples may observe intermediate cleavage products of 19 kDa), and the approximately 12 kDa cleaved form.
Caspase-3 is only cleaved during apoptosis, so in most cases, the caspase-3 precursor protein can be detected without drug treatment. If you want to detect cleaved caspase-3, we strongly recommend inducing the samples into the apoptosis pathway (e.g., using staurosporine-treated samples as a positive control for drug administration), and at the same time, a decrease in the caspase-3 precursor protein content will be observed after induction.
Please note that different antibodies may detect different forms of caspase-3 protein: for example, ab32499 can only detect the precursor protein, ab32042 can only detect the 17 kDa cleaved form, ab32351 can detect both the precursor protein and the 17 kDa cleaved form, and ab179517 can detect both the precursor protein and the 12 kDa cleaved form.
When using the same antibody to detect samples from different species, there may also be differences in the forms of caspase-3 protein detected: for example, ab184787 can detect both the precursor and cleaved forms of caspase-3 in human samples (e.g., Jurkat), but cannot detect the cleaved form of caspase-3 in mouse and rat samples.
Due to the presence of multiple post-translational modifications of caspase-3, the size of the bands detected in WB may differ from the predicted values.

Positive control

Caspase-3 precursor: HAP1, Jurkat, HeLa whole cell lysate
Caspase-3 cleaved form: Staurosporine-treated HAP1, Jurkat, HeLa whole cell lysate (see product datasheet for details)

Negative control

Human CASP3 (Caspase-3) KO HAP1 cell line/whole cell lysate

Example results

WB-Anti-pro Caspase-3 antibody [E83-103] (ab32499).

Figure 3: WB-Anti-pro Caspase-3 antibody [E83-103] (ab32499).

Lane 1: HAP1 whole cell lysate
Lane 2: HAP1 whole cell lysate + staurosporine (1 μM for 4h)
Lane 3: Caspase-3 knockout HAP1 whole cell lysate
Lane 4: Caspase-3 knockout HAP1 whole cell lysate + staurosporine (1 μM for 4h)
Green band size: 32 kDa (ab32499, pro Caspase-3)
Red band size: 37 kDa (ab8245, GAPDH)

WB- Anti-Cleaved Caspase-3 antibody [E83-77] (ab32042)

Figure 4: WB- Anti-Cleaved Caspase-3 antibody [E83-77] (ab32042).

Lane 1: HAP1 whole cell lysate + DMSO (24h)
Lane 2: HAP1 whole cell lysate + Starvation (2 μM for 24h)
Lane 3: Caspase-3 knockout HAP1 whole cell lysate + DMSO (24h)
Lane 4: Caspase-3 knockout HAP1 whole cell lysate + Starvation (2 μM for 24h)
Lane 5: Hela whole cell lysate + DMSO (24h)
Lane 6: Hela whole cell lysate + Starvation (2 μM for 24h)

Green band size: 17 kDa (ab32042, Cleaved Caspase-3)
Red band size: 130 kDa (ab130007, Vinculin)

WB-Anti-Caspase-3 antibody [EPR18297] (ab184787)

Figure 5: WB-Anti-Caspase-3 antibody [EPR18297] (ab184787).

Lane 1: Untreated Jurkat whole cell lysate.
Lane 2: Jurkat whole cell lysate treated with 1 μM staurosporine for 4 hours.

Predicted band size: 32 kDa.
Detected band sizes: 17, 32 kDa.

WB-Anti-Cleaved Caspase-3 antibody [EPR21032] (ab214430)

Figure 6: WB-Anti-Cleaved Caspase-3 antibody [EPR21032] (ab214430).

Lane 1: NIH/3T3 whole cell lysate
Lane 2: NIH/3T3 whole cell lysate + staurosporine (1 μM for 4h)

Predicted band size: 31 kDa
Detected band sizes: 17, 19, 24, 29, 32 kDa

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

Add a complex protease inhibitor to avoid degradation of the target protein.
Keep the sample on ice throughout the sample preparation process.
Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.
Sample sonication can enrich nuclear proteins and enhance WB signal intensity.

Electrophoresis:

For target proteins with smaller molecular weights (such as molecular weight <25 kDa), use a 15% separation gel for electrophoresis.
Load at least 20 μg total protein for electrophoresis.

Transfer:

For target proteins with smaller molecular weights, it is recommended to use a 0.22 μm PVDF membrane.
We recommend using Ponceau S staining after transfer to confirm the success of the transfer (if using fluorescent labeling detection, make sure the Ponceau S is completely washed off).

Antibody incubation:

Please choose the optimal antibody working concentration according to the product manual.