What does proteinase K do what does SDS do?
Proteinase K is used for the destruction of proteins in cell lysates (tissue, cell culture cells) and for the release of nucleic acids, since it very effectively inactivates DNases and RNases. The enzyme’s activity towards native proteins is stimulated by denaturants such as SDS.
What temperature is proteinase K activity?
between 20 and 60°C
Proteinase K is active in a wide range of temperatures and buffers with optimal activity between 20 and 60°C and a pH between 7.5 and 12.0 (1, 2). Activity is stimulated when up to 2% SDS or up to 4 M urea are included in the reaction (3).
What does proteinase K do in RNA extraction?
Proteinase K is critical because it digests proteins eliminating contamination from nucleic acid preparations, in addition to inactivating the nucleases that could degrade DNA or RNA during purification.
Why do you need to inactivate proteinase K?
For example, in the nucleic acid extraction protocol, proteinase K is added to cell lysate and then an incubation period follows to ensure a complete digestion. To prevent potential digestion of your samples, proteinase K is inactivated after incubation.
What does proteinase K cleave?
Proteinase K cleaves peptide bonds next to the carboxyl group of N-substituted hydrophobic, aliphatic, and aromatic amino acids. It also cleaves peptide amides.
How do you make proteinase K?
Proteinase K is prepared commonly as a 20 mg/ml stock solution in sterile water (stable for 1 year at −20°) or in a solution of 50 mM Tris, pH 8.0, 1 mM CaCl2 (stable for months at 4°).
How is proteinase K produced?
Proteinase K, produced by the fungus Tritirachium album Limber, is a serine protease that exhibits a very broad cleavage specificity. It cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples.
How do you make Proteinase K?
Is Proteinase K necessary for DNA extraction?
During the extraction of DNA (or nucleic acids in general), there is a lot of contaminating proteins present. The addition of proteinase K degrades these nucleases and protects the nucleic acids from nuclease attack. In addition, proteinase K is stable over a wide pH range and is well suited for use in DNA extraction.
What is proteinase K used for?
Proteinase K is commonly used in molecular biology to digest protein and remove contamination from preparations of nucleic acid. Addition of proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification.
How is Proteinase K produced?
What is Proteinase K made of?
Proteinase K is a typical member of the subtilisin family of proteinases (S8). The amino acid sequence has been derived by Edman degradation [2] and from the gene sequence [3]; the polypeptide chain consists of 278 amino acids, with molecular mass 28 930, and the pI is 8.9 [1].