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.
What is proteinase K biology?
Proteinase K is a subtilisin-related serine protease that hydrolyzes a variety of peptide bonds and is frequently used to cleanup enzymatic reactions or cell lysates. Highly characterized for more consistent performance. Optimal activity and stability for up to 24 months.
Where does proteinase cleave?
Where 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.
What is the role of proteinase K 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.
What is detergent in DNA extraction?
To get the DNA from a cell, scientists typically rely on one of many DNA extraction kits available from biotechnology companies. During a DNA extraction, a detergent will cause the cell to pop open, or lyse, so that the DNA is released into solution. Then alcohol added to the solution causes the DNA to precipitate out.
How does serine protease work?
Serine proteases catalyze peptide bond hydrolysis in two sequential steps. In the first (acylation) reaction, the nucleophilic serine attacks the substrate scissile bond, forming first a tetrahedral intermediate and then a covalent acyl-enzyme with release of the C-terminal fragment.
How do you make Proteinase K?
To ensure maximum Proteinase K activity, use water that has been treated with 0.1% DEPC (DEPC, GoldBio Catalog # D-340 [CAS 1609-47-8, mw. = 162.14 g/mol]) to make the buffer. Add 1 ml of DEPC to 1 L of H2O, shake vigorously, and let incubate for 12 hours at 37°C to inactivate any RNases.
What is SDS in DNA extraction?
SDS provides a negative charge to each protein as a function of their size. Accordingly, all of proteins have the same shape in the gel separation they are separated only for their size. Furthermore, SDS can be used to aid in lysing cell during DNA extraction.
What is K protocol?
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.
Why do we neutralize the proteinase K after incubation?
To prevent potential digestion of your samples, proteinase K is inactivated after incubation. The common temperature for inactivation is 95°C. Even in the typical mouse-tail protocol, proteinase K is regularly used to inhibit harmful nucleases.
What is the difference between protease and proteinase K?
The key difference between proteinase K and protease is that proteinase K is useful in digesting proteins and removing contamination from nucleic acid preparations, whereas protease is useful in biological functions such as digestion of ingested proteins, protein catabolism, and cell signaling.