One article to solve these problems encountered by proteinase K
1. What is proteinase K?
Proteinase K is an endopeptidase and belongs to the subtilisin group of serine proteases. In molecular biology laboratories, proteinase K is useful in the preparation of DNA or RNA samples by degrading and inactivating proteins.
2. What is the appropriate temperature for proteinase K activation?
Proteinase K activity increases with temperature (up to a certain point). The suitable temperature range is between 50-65˚C. Higher temperatures help proteins unfold, thereby reducing the ability of proteinase K to break down these proteins.
3. How does proteinase K participate in cell lysis?
When it comes to cell lysis, especially downstream DNA isolation and purification, proteinase K can digest surface proteins as part of the lysis step. Going further into this procedure, when it is necessary to resuspend and lyse the nucleus in a buffer containing proteinase K, proteinase K will help digest proteins that would otherwise degrade the sample.
4. How is proteinase K used for mammalian DNA isolation?
The first step in isolating DNA from mammalian cells and leukocytes is to lyse the cells and spin them to collect the pellet. After suspension, detergent (SDS) is added to destroy the nuclear membrane. After proteinase K digests the protein, both histone and non-histone proteins release DNA. In addition, proteinase K digests and inactivates ribonuclease in solution.
5. Can proteinase K be used for RNA isolation?
In the RNA isolation process, the addition of proteinase K will degrade and inactivate even a trace amount of ribonuclease in the presence of the substrate. The additional phenol, chloroform and isopropanol precipitation steps reduce protein contamination and make the final precipitate easier to re-dissolve.
6. What is the relationship between proteinase K and calcium?
Proteinase K binds to two Ca2+ ions, which helps maintain the stability of the enzyme, especially at elevated temperatures. Calcium can also protect proteinase K from autolysis. Although calcium helps maintain the thermal stability of proteinase K, it is not necessary for proteolytic activity.
7. Will EDTA inactivate proteinase K?
Chelating agents such as EDTA or EGTA have no direct effect on the enzyme activity of proteinase K. The reason for using EDTA and proteinase K in DNA or RNA purification is to remove calcium. However, because calcium is related to the stability of proteinase K, the addition of EDTA will affect calcium, thereby reducing the activity of proteinase K to a certain extent.
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