What is proteinase K made of?
Proteinase K is known enzyme belonging to the class of serine proteases that cleave adjacent to the carboxylic group peptide bonds of aromatic and aliphatic amino acids. It’s also exhibited a very broad cleavage specificity. The enzyme was discovered in extracts of the fungus Engyodontium album.
How does proteinase K work?
In molecular biology, main application of proteinase K is to digest proteins during nucleic acids purification in order to remove protein contaminants:
– Proteinase K quickly inactivates nucleases (enzymes that digest nucleic acids) that degrade DNA or RNA molecules during the cleansing.
– Proteinase K is activated by calcium. It does not affect the activity of the proteinase, but protects again autolysis, improves thermal stability, and regulates substrate binding capacity. To increase the stability of Proteinase K, thereby enhancing its action, Urea as well as SDS (sodium dodecyl sulfate) or elevated temperature at 37-60C is commonly used. Inactivation of Proteinase K is occurring at temperature above 65C.
What is the purpose of Proteinases?
Proteinase K is used to break down proteins in cell lysates (tissues, cell culture cells) and to release nucleic acids due to its action described above. Proteinase K application purpose:
DNA isolation from various tissues;
Removal of DNAses and RNAses: Proteinase K and RNases can act together in the lysis buffer to degrades contaminating RNAs and proteins;
Increasing cloning efficiency;
Stable in a wide range of pH 4-12.5;
Automated nucleic acids isolation;
Why is proteinase K used in DNA extraction?
During DNA extraction, many contaminating proteins are present. Proteinase K is used in many DNA isolation protocols and commercial kits to cleave these contaminating proteins. In addition, proteinase K destroys nucleases and protects nucleic acids from their attacks.
How is proteinase K prepared for DNA isolation?
Proteinase K is available as lyophilized powder or as liquid. To ensure maximum Proteinase K activity, use double distillate water with presence of 0.1% DEPC or dissolve in 50 mM Tris-HCl, pH = 7.5-8.0, 1-5 mM Ca2+ (calcium chloride, calcium acetate) and 50% glycerol required for long-term storage. Invitrogen suggested working concentration of Proteinase K is 0.05 to 1 mg/mL, moreover, enzyme activity can be stimulated by 0.2 to 1% SDS or by 1 to 4M urea.
How much proteinase K to use for DNA extraction?
For the DNA extraction, 200μg/ml of proteinase K working solution is sufficient. Generally, the proteinase K provided in 20mg/ml concentrations as recommended by the TermoFisher manufacturer. So 10μL of proteinase K (from the 20mg/ml stock) can be added to the 990μL purified water, the working solution becomes 200μg/ml. For optimal proteinase action, it is either incubated at 50-55 degrees before addition or after addition along with sample and buffer.