Cell Function
Analysis and Solutions to Common Issues in Caspase Enzyme Activity Detection
Source: Elabscience®Published: Jul 23,2024
After discussing the principles and classifications of programmed cell death in our previous session, today, we will provide a brief summary of the three main types: apoptosis, necrosis, and pyroptosis.
We will focus on introducing the caspase family's significant role in apoptosis and pyroptosis processes, along with addressing common issues and solutions in caspase enzyme activity detection using spectrophotometry.
Let's start with a brief review of the involvement of the caspase protease family in apoptosis and pyroptosis:

Next, let's delve into the members of the Caspases protease family. Caspases, also known as cysteine-aspartic proteases, play a pivotal role as key regulatory elements in the programmed cell death signaling pathway.
The process of apoptosis essentially involves the activation of caspases, triggering a cascade reaction that exerts the enzymatic cleavage function of apoptotic proteases. The caspases protease family is present in the cytoplasm, exhibiting high homology and structural similarity. All members contain a cysteine activation site, selectively cleaving peptide bonds after aspartic acid residues. Currently, 14 types of caspases have been discovered. In mammals, the caspases protease family can be classified into three major categories based on their structure and function:

Caspases enzymes are often present in cells in the form of proenzymes, lacking cleavage activity. They exist in the form of cleavage bodies after activation. This article focuses on the method of detecting caspases activity using an enzyme-linked immunosorbent assay (ELISA) with caspases activity assay kits (spectrophotometry). The principle is mainly based on the fact that caspases can catalyze the specific substrate Ac-XXXD-pNA to produce yellow pNA (p-nitroaniline). pNA has a strong absorption near 405 nm, allowing the detection of caspases activity by measuring absorbance.
The main schematic diagram is as follows:

The detection process of the caspases enzyme activity assay kit (absorbance method) mainly includes three major blocks: sample pretreatment, sample lysis, and enzyme activity detection. For this detection process, the following common issues and solutions are summarized:
Issues and Solutions in the Sample Pretreatment Process:

Issues and Solutions in the Sample Lysis Process:

Issues and Solutions in the Sample Detection Process:

Issues and Solutions Due to Other Factors:

In conclusion, attention to detail determines success. By meticulously controlling the details of each experimental step, caspases enzyme activity detection (absorbance method) can be foolproof, ensuring the most accurate experimental data.