Storage For long time storage, store the Proteinase K powder at 4ºC and dilution buffer at -20℃ separately. For use, dissolved solution should be stored at -20ºC.
Specific Activity: 40 U/mg protein Form: Contains 100 mg Proteinase K, Lyophilized Powder
Features Active in a wide range of reaction products
Description Proteinase K is an endolytic protease that cleaves peptide bonds at the carboxylic sides of aliphatic, aromatic or hydrophobic amino acids. The Proteinase K is classified as a serine protease. The smallest peptide to be hydrolyzed by this enzyme is a tetrapeptide.
Applications Isolation of genomic DNA from cultured cells and tissues Removal of DNases and RNases when isolating DNA and RNA from tissues or cell lines Determination of enzyme localization Improving cloning efficiency of PCR products
Quality Control DNase Activity: None detectable enzyme activity with λ DNA after 6 hrs incubation at 37ºC. RNase Activity: None detectable ribonuclease activity after 16 hrs incubation at 25ºC.
Source Pichia pastoris cells with a cloned gene encoding Tritirachium album endolytic protease (Proteinase K).
Molecular Weight 28.9 kDa monomer (6).
Dilution Buffer
50 mM Tris-HCl (pH 7.5), containing 5 mM calcium chloride and 50% (v/v) glycerol. .
Definition of Activity Unit One unit of the enzyme liberates Folin-positive amino acids and peptides corresponding to 1 µmol tyrosine in 1 min at 37°C using denatured hemoglobin as the substrate.Enzyme activity is assayed in the following mixture: 0.08 M potassium phosphate (pH 7.5), 5 M urea, 4 mM NaCl, 3 mM CaCl2 and 16.7 mg/ml hemoglobin.
Inhibition and Inactivation
Inhibitors: Proteinase K is not inactivated by metal chelators, by thiol-reactive reagents or by specific trypsin and chymotrypsin inhibitors. Phenylmethylsulfonyl fluoride and diisopropyl phosphorofluoridate completely inhibit the enzyme.
Inactivated by heating at 95°C for 10 minutes.
Note
Optimum activity at 50-55°C.
Rapid denaturation of enzyme occurs at temperatures above 65°C.
The recommended working concentration for Proteinase K is 0.05-1 mg/ml. The activity of the enzyme is stimulated by 0.2-1% SDS or by 1-4 M urea.
Ca+2 protects Proteinase K against autolysis, increases the thermal stability, and has a regulatory function for the substrate binding site of Proteinase K.
Stable over a wide pH range: 4.0-12.5, optimum pH = 7.5-8.0.
