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Analysis of common problems and solutions for Caspases activity assay in the flow classroom

Time：

2023-05-16

　　In this article we will give a brief summary of the three types of programmed death, i.e. apoptosis, cell necrosis, and cell scorching, and focus on the Caspase family, which plays an important role in the process of apoptosis and scorching, as well as common problems and solutions for Caspase enzyme activity assays (spectrophotometric methods).

　　Let's first briefly review the Caspases white enzyme family involved in apoptosis and scorch wide related content-

　　Next, let's learn about the members of the Caspases family of proteases, which play a pivotal role as key regulatory links in the programmed cell death signaling pathway.

　　The process of apoptosis is actually a cascade reaction in which Caspases are activated and perform the enzymatic cleavage function of apoptotic proteases. caspases protease family exists in the cytosol, they are highly homologous, structurally similar, and all contain a cysteine activation site that specifically breaks the peptide bond after the aspartate residue. 14 species of Caspases have been identified, mammalian The Caspases family of white enzymes can be divided into three main groups based on structure and function.

　Casnasespe cells often exist in the form of zymogens, which do not have immediate-cutting activity, but exist in the form of precursors after being activated. This paper introduces the method of detecting Caspases active bodies with the Sea Standard Pass Caspases Activity Assay Kit (spectrophotometric method). pNA (p-nitroaniline), which has a strong absorption near 405 nm, so that the activity of Caspases can be detected by measuring the absorbance.

　　The main schematic diagram is shown below.

　　The detection process of Caspase Sex Detection Kit (absorbance method) mainly includes sample pretreatment, sample lysis and enzyme activity detection, for which the following common problems and solutions are summarized.

　　Problems and solutions in the sample pre-treatment process

Problems and solutions in the sample lysis process

其他因素引起的问题和解决办法

Problems and solutions in the sample testing process

其他因素引起的问题和解决办法

Problems caused by other factors and solutions

　　To sum up, details determine success or failure, and the Caspases activity assay (absorbance assay) can be performed without fail to obtain the most accurate experimental data if the details of each experimental step are controlled in place.