Description
Proteinase K (also Protease K, Endopeptidase K, Peptidase K or Tritirachium  Alkaline Phosphatase) (EC 3.4.21.64) is a non-specific, broad spectrum serine protease that is isolated from the saprophytic fungus Tritirachium album. It is not activated by metal ions, chelating agents (for example, EDTA), sulfhydryl reagents, or by trypsin or chymotrypsin inhibitors. It is stable over a wide pH range (4–12.5), with optimal activity at pH 6.5–9.5. Activity can be stimulated by addition of denaturing agents (SDS and urea). The temperature optimum for the enzyme is 65°C (twelve times more active at 65°C than at 25°C). Rapid denaturation of the enzyme occurs at temperatures above 65°C.

Application
Proteinase K is used to rapidly inactivate endogenous nucleases such as RNases and DNases when isolating RNA or DNA from tissues and cell lines, such as bovine endometrial epithelial cells for herpes viral DNA extraction, and viral RNA extraction from nasal swabs. This enzyme can also be used as a component of phase lock and direct PCR lysis buffer to remove nucleases in the preparation of tissue sections for in situ hybridization.

Source: saprophytic fungus Tritirachium album

Proteinase K, Solution
Proteinase K solution is an optimized, 20 mg/ml solution that is free from all nuclease contamination. The specific activity is >30 units/mg at 37°C. The buffering conditions used ensure that the enzyme retains its full activity and offers convenience to the researchers. Proteinase K solution is stable for one year if stored properly.

Proteinase K, Lyophilized Powder
The dry, lyophilized powder is highly soluble (>50 mg/ml); however, stock solutions are normally 20 mg/ml. The proteinase K is completely RNase and DNase free and has a specific activity of >30 units/mg at 37°C. The recommended storage buffer is 20mM Tris-HCl (pH7.4), 1mM CaCl2, 50% glycerol. Proteinase K is stable for one year in this storage buffer and recommended storage is –20°C.

Reconstitute Proteinase K
Dissolve Proteinase K:
• Dissolve in 10 mM Tris HCl, pH 7.5, 20 mM calcium chloride, and 50% glycerol, then store at –20°C.
• Dissolve in 50 mM Tris HCl (pH 8.0), 1–5 mM calcium acetate, then store at 2°C–8°C.
Storage at –20°C in the absence of glycerol can lead to precipitation of the Proteinase K. Bacterial growth can occur in solutions stored at 2°C–8°C over extended periods of time. Ca2+ can serve as a stabilizer to suppress autolysis.

Storage Buffer (not included): 50 mM Tris, pH 8, 3 mM CaCl2, 50% Glycerol

Storage: Store at –20°C.  Avoid multiple freeze-thaw cycles.

Unit Definition: One unit liberates 1 µmol of Folin-positive amino acids, measured as tyrosine, at 37°C, pH 7.5, using denatured bovine hemoglobin as the substrate.