Hello everyone,
We are planning to use this protocol for our cardiomyocyte isolation: https://www.jove.com/pdf-materials/4205/isolation-of-cardiomyocyte-nuclei-from-postmortem-tissue
I am just curious about the functions of some reagents they have in the lysis buffer. Can you please help me to clarify the function of DTT and EGTA added in the buffer?
Thank you so much!
Hello Nguyen Nguyen
DTT is a reducing agent that reduces disulfide bonds and protects from oxidation damage. DTT at lower concentration (0.5mM) stabilize enzymes and other proteins which possess free sulfhydryl group and restore its activity.
Please note that DTT may be added to lysis buffer when the protein of interest in the experiment is redox-sensitive but should be left out in other cases since it has the potential to cause loss of structure and activity as a result of the broken disulfide bonds.
EGTA is an inhibitor of metalloproteases. Similar to EDTA, EGTA is a metal chelator. It will chelate divalent cations which are cofactors of many enzymes such as DNases and proteases. By chelating the co-factors of these enzymes, the activity of these enzymes decreases, as they wouldn’t be available for the reaction. However, EGTA has a lower affinity for chelating Mg2+ ions compared to EDTA. I presume that since Mg2+ will be present in the buffer, adding EGTA would be more preferable than adding EDTA.
Best.
DTT will break disulfide bonds as it is a potent reducing agent, and EGTA is a chelator that binds to mostly divalent cations, especially calcium ions, and abrogate their movement into the cells.