Proteinase K (also protease K, endopeptidase K, peptidase K or Tritirachium alkaline phosphatase) (EC 3.4.21.64) is a non specifc, broad spectrum serine protease that is isolated from the saprophytic fungus Tritirachium album.

Proteinase K is routinely used for the purification of target material from contaminating proteins, for the isolation of RNA and DNA and to inactivate other protein enzymatic activities. Proteinase K exhibits high activity in the presence of SDS, urea and EDTA. This allows the use of EDTA in the purification of DNA or RNA to inhibit calcium dependent nucleases.

The dry, lyophilised powder is highly soluble (>50 mg/ml), however stock solutions are normally 20 mg/ml. The Proteinase K is completely free from RNase and DNase and has a specific activity of >30 units/mg at 37 °C. The recommended storage buffer is 20 mM Tris-HCl (pH 7,4), 1 mM CaCl₂, 50% glycerol. Proteinase K is stable for 1 year in this storage buffer, recommended storage is −20 °C.