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Caspase 3/7 Activity Assay Kit (Colorimetric Method)
SKU: E-CK-A383-100
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Caspase 3/7 Activity Assay Kit (Colorimetric Method)
Introduction
Elabscience® Caspase 3/7 Activity Assay Kit uses spectrophotometry to detect the caspase 3/7 activity of cells, tissue lysates or other samples.
Product Details
Properties
| Application | Caspase |
| Assay type | Qualitative or quantitative |
| Detection method | Colorimetric Method |
| Sample type | Cell and tissue samples |
| Assay time | 4-6 hours |
| Detection instrument | Spectrophotometer |
| Other reagents required | PBS(E-BC-R187)，Bradford Protein Determination Kit(E-BC-K168-S or E-BC-K168-M) |
| Storage | -20°C, shading light |
| Shipping | Ice bag |
| Expiration date | 12 months |
Detection Principle
Caspase 3, also known as CPP32, Yama or apopain, belongs to the CED-3 subfamily of the caspase family. It is a key enzyme in the process of apoptosis and the most studied caspase in mammalian cells. Caspase 3 exists in the cytoplasm in the form of zymogen under normal condition and has no activity. In the stage of apoptosis, caspase 3 is activated, and the activated caspase 3 consists of two large subunits and two small subunits, which cleaves the corresponding cytoplasmic and nuclear substrates and ultimately leads to apoptosis. This caspase 3/7 activity assay kit is based on caspase 3/7 that can catalyze the substrate Ac-DEVD-pNA to generate yellow pNA (p-Nitroaniline). The pNA has a strong absorption near 405 nm, The activity of caspase 3/7 can be calculated by determining the absorbance at 405 nm.
Storage
Store at -20°C for 1 year. It is recommended to aliquot the AcDEVD-pNA (4 mM) into smaller quantities and store in the dark. Avoid repeated freezing and thawing.
Notes
- This kit is for research use only.
- Please take safety precautions and follow the procedures of laboratory reagent operation.
- The pNA (4-nitroaniline) is toxic to the human body. Please be careful during operating, and pay attention to avoid direct contact with the human body or inhalation. The pNA will solidify at a lower temperature and stick to the bottom, wall or cap of centrifuge tube. It can be used until it is completely melted in a water bath at 20~25°C for a moment.
- When the level of activated caspase in the sample is low, first confirm whether apoptosis is obvious. If cell apoptosis is obvious and it is confirmed that the caspase can be activated, the time of inducing cells can be adjusted properly to find a time point when the caspase activation is relatively strong, so as to detect the activation of the caspase. It is recommended to plot a time curve, such as 0, 2, 4, 8, 16, and 24 hours of induction, or 0, 1, 2, 4, 8, and 16 hours, or 0, 1, 2, 4, 6, and 8 hours. Specific induction time needs to be determined according to the specific situation.
- In the reaction system of this kit, the initial concentration of the substrate is 0.2 mM. For most samples, the substrate is saturated within 2 hours of incubation at 37°C. For a few samples that the enzyme vitality is particularly high, it is necessary to use Cell Lysis Buffer to dilute the sample appropriately before determination.