BLIRT Proteinase K

Mutagen and recombinant Proteinase K from Tritirachium album is non-specific, subtilisin-related serine protease with a very high specific activity.

Name Part Number Pack Size
Proteinase K RP100B 100 mg
  RP101B 250 mg
  RP102B 1000 mg

 

Mutagen and recombinant Proteinase K from Tritirachium album is non-specific, subtilisin-related serine protease [1] with a very high specific activity. The enzyme cleaves peptide bonds at the carboxylic sides of aliphatic, aromatic or hydrophobic amino acids. The smallest peptide to be hydrolyzed by this enzyme is a tetrapeptide. Proteinase K is supplied as a lyophilized powder.

Properties

- specific activity: approx. 30 u/mg

- stable over a wide pH range: 4.0-12.5, optimum pH 7.5-8.0

- not inactivated by metal chelators, by thiol-reactive reagents or by specific trypsin and chymotrypsin inhibitors

- the activity of the enzyme is stimulated by 0.2-1% SDS or by 1-4 M urea

- Ca2+ protects Proteinase K against autolysis, increases the thermal stability and has a regulatory function for the substrate binding site of Proteinase K [6]

Applications

- purification of target material from contaminating proteins

- isolation of genomic DNA and mRNA from cultured cells [3]

- removal of DNases and RNases when isolating DNA and RNA from tissues or cell lines [2,3]

- determination of enzyme localization [4]

- improving cloning efficiency of PCR products [5]

References

1. Ebeling, W., et al., Proteinase K from Tritirachium album Limber, Eur. J. Biochem., 47, 91-97, 1974.

2. Wiegers, U., Hilz, H., A new method using ‘proteinase K’ to prevent mRNA degradation during isolation from HeLa cells, Biochem. and Biophys. Res. Commun., 44, 513-519, 1971.

3. Hilz, H., et al., Stimulation of proteinase K action by denaturing agents: application to the isolation of nucleic acids and the degradation of “masked” proteins, Eur. J. Biochem., 56, 103-108, 1975.

4. Brdiczka, D., Krebs, W., Localization of enzymes by means of proteases, Biochim. Biophys. Acta, 297, 203-212, 1973.

5. Crowe, J.S., et al., Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion, Nucleic Acids Res., 19, 184, 1991.

6. Bajorath, J, et al, The enzymatic activity of proteinase K is controled by calcium., Eur. J. Biochem., 176, 441-447, 1988.

Storage Temperature

Lyophilized powder storage at -20ºC.

Recommended Reaction and Storage Buffers

- 50 mM Tris-HCl (pH 8.0), 5 mM CaCl2 and 50% (v/v) glycerol (storage at -20ºC)

- 50 mM Tris-HCl (pH 8.0), 5 mM CaCl2 (storage at +4ºC)

Inhibitors       

Phenylmethylsulfonyl fluoride (PMSF) and diisopropyl phosphorofluoridate (DPF) completely inhibit the enzyme. Proteinase K is also inactivated by heating above 65ºC for 20 min.

Quality control

The absence of exonucleases, ribonucleases, and nucleic acids confirmed by appropriate quality tests.

Unit definition

One unit of the enzyme liberates Folin-positive amino acids and peptides corresponding to 1 µmol tyrosine in 1 min.

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