Why do we add protease inhibitors when extracting tissue proteins to prevent protein denaturation, and then add SDS to break down proteins during sample preparation?

During protein extraction and sample preparation, the addition of protease inhibitors and subsequent addition of SDS serve different experimental purposes. Although these two steps seem contradictory, they each have clear scientific significance.

 

1. Purpose of Adding Protease Inhibitors

During tissue disruption and protein extraction, intracellular proteases may become activated. These proteases can begin to degrade proteins within the cells, leading to the destruction or degradation of target proteins. This can affect experimental results, especially in downstream analyses such as protein quantification and immunoblotting. Therefore, adding protease inhibitors during protein extraction is to prevent the activity of these proteases and protect target proteins from degradation, ensuring that the extracted proteins are as complete and intact as possible.

 

2. Purpose of Adding SDS

In subsequent sample preparation, the purpose of adding SDS is to achieve complete denaturation of proteins, especially in SDS-PAGE. The specific functions include:

1. Disruption of Protein's Native Structure: SDS is a potent detergent that can disrupt the secondary, tertiary, and quaternary structures of proteins, linearizing them.

2. Imparting Negative Charge: SDS binds to proteins, allowing them to be separated based on size rather than charge.

3. Standardizing Electrophoretic Mobility: Once linearized, the migration rate of proteins primarily depends on molecular weight, facilitating quantification and separation.

 

In summary, protease inhibitors are mainly used to prevent protein degradation during extraction, while SDS is used to denature proteins for downstream analysis.

 

Bio-Techne Corporation - Characterization of Biological Products, High-Quality Multi-Omics Mass Spectrometry Detection Service Provider

 

Related Services:

Sample Preparation