How do you heat inactivate Proteinase K?
While the activity of proteinase K increases with temperature, and is optimized at about 65 ˚C, heating proteinase K to 95 ˚C for 10 minutes will inactivate it.
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.
How do you resuspend Proteinase K?
(20 mg/ml) Purchase as a lyophilized powder and dissolve at a concentration of 20 mg/ml in sterile 50 mM Tris (pH 8.0), 1.5 mM calcium acetate. Divide the stock solution into small aliquots and store at -20°C. Each aliquot can be thawed and refrozen several times but should then be discarded.
What is the optimal temperature for Proteinase K?
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).
Is Proteinase K Stable at room temperature?
QIAGEN Proteinase K is stable for up to 1 year after delivery when stored at room temperature. To prolong the shelf-life of Proteinase K, storage at 2–8°C is recommended.
Can Proteinase K degrade DNA?
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.
Is proteinase K Stable at room temperature?
Does SDS inhibit proteinase K?
Proteinase K will not be inhibited by Guanidinium chloride, Guanidinium thiocyanate, urea, Sarkosyl, Triton X-100, Tween 20, SDS, citrate, iodoacetic acid, EDTA or by other serine protease inhibitors like Nα-Tosyl-Lys Chloromethyl Ketone (TLCK) and Nα-Tosyl-Phe Chloromethyl Ketone (TPCK).
How does EDTA inactivate DNases?
EDTA does not inactivate DNase I, it just removes the Mg ion so that the DNase I no longer active due to lack of Mg. You need to add more EDTA than the amount of Mg ions present to inactivate. If you subsequently (without purification) tried to use a different enzyme that was also Mg dependent, it would fail to work.
What was the purpose of the proteinase K?
Proteinase K is used during DNA extraction to digest many contaminating proteins present. It also degrades nucleases that may be present in DNA extraction and protects the nucleic acids from nuclease attack.
Does proteinase K digest itself?
A protein that digests proteins, but does not get digested by itself.
What does K stand for in proteinase K?
The enzyme was discovered in 1974 in extracts of the fungus Engyodontium album (formerly Tritirachium album). Proteinase K is able to digest hair (keratin), hence, the name “Proteinase K”.
Can a QIAGEN protease be inactivated by heat?
QIAGEN Protease is inactivated by incubation at 70°C for 15 minutes. To our knowledge, Proteinase K cannot be completely heat-inactivated. Even when incubating at 95°C for 10 minutes, some enzymatic activity remains.
Can a Proteinase K wash be heat inactivated?
To our knowledge, Proteinase K cannot be completely heat-inactivated. Even when incubating at 95°C for 10 minutes, some enzymatic activity remains. This will not negatively affect the QIAamp Procedure, since the enzyme will be efficiently removed by the wash steps in the protocols.
What kind of activity does QIAGEN proteinase K have?
QIAGEN Proteinase K is a subtilisin-type protease, which cleaves at the carboxyl side of hydrophobic, aliphatic and aromatic amino acids. It is particularly suitable for short digestion times. It possesses a high specific activity over a wide range of temperatures and pH values with substantially increased activity at higher temperature.
Which is the best alternative to proteinase K?
Proteinase K is supplied in the following QIAGEN kits: QIAGEN Protease is a serine protease isolated from a recombinant Bacillus strain and is an economical alternative to Proteinase K for isolation of native DNA and RNA from a variety of sources. QIAGEN Protease is completely free of DNase and RNase activities.