CNMCS Kit: Extracting from Tissues. Due to the uneven concentration of proteins across areas, it’s important to choose tissues that would have a higher distribution of proteins for maximum extraction. The initial step when extracting from tissue is to chop the tissue and homogenize it before lysis. This mechanical disturbance breaks the tissue surface and structure down. After centrifuging the tissue, the first protein available is cytoplasmic. Next, using chemical buffers at ice-cold temperatures and further centrifugation, nuclear proteins become available. More chemical buffer is added, and the third centrifuge rotation provides membrane proteins. Finally, with the aid of additional buffer, the remaining pellet can be centrifuged a final time for cytoskeleton proteins.
CNMCS: Extracting from Culture Cells. This method is different than extracting from tissues since cells only require lysis of the external membrane to extract proteins, which reduces some steps. First, the culture cells are centrifuged with ice-cold buffer, then passed through a needle base to disrupt the cell membrane and release cell nuclei. A microscope should be used to monitor the level of disruption. Next, spin the cell mix to release cytoplasmic proteins. The cell pellet is suspended back in ice-cold buffer and centrifuged multiple times to release nuclear proteins. Using a different buffer and centrifuging, membrane proteins are available. Finally, using warm buffer and rotating at room temperature releases the cytoskeleton proteins.
Simplifying Challenges in Protein Extraction. The extraction of proteins is a challenging process that often requires management for precision. Often during the cell lysis process, proteins may become denatured from chemical buffers, and this may result in useless proteins that fail to refold or fold into incorrect shapes. The presence of proteinases may also cause digestion of protein; this needs to be accounted for during purification. Cross contamination also tends to be a significant issue. The BioChain CNMCS Kit is carefully designed and quality tested to avoid these issues, with detailed troubleshooting included in the manual.