Proteinase K (recombinant), PCR grade
#EO0491 1 ml (~600-1000 u/ml, ~14-22 mg/ml) #EO0492 5x1 ml (~600-1000 u/ml, ~14-22 mg/ml) Related Documents (in pdf, ~40-60 KB):
Certificate of Analysis: #EO0491, #EO0492
MSDS (English)
MSDS (English-USA)
MSDS (German)
Features
- Ready-to-use solution.
- 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 (1). The smallest peptide to be hydrolyzed by this enzyme is a tetrapeptide.Applications
- Isolation of genomic DNA from mouse tail, see Purification of Genomic DNA from Mouse Tail with Proteinase K.
- Isolation of genomic DNA from cultured cells, see Protocol for Purification of DNA from Cultured Eukaryotic Cells with Proteinase K.
- 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).
Quality Control
The absence of endo-, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests.Concentration
Approx. 600-1000 u/ml, 14-22 mg/mlSource
Pichia pastoris cells with a cloned gene encoding Tritirachium album endolytic protease (Proteinase K).Molecular Weight
28.9 kDa monomer (6).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 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.Storage Buffer
The enzyme is supplied in: 10 mM Tris-HCl (pH 7.5), containing calcium acetate and 50% (v/v) glycerol.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 (1).
- 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 14 M urea (3).
- Ca2+ protects Proteinase K against autolysis, increases the thermal stability and has a regulatory function for the substrate binding site of Proteinase K (7).
- Stable over a wide pH range: 4.0-12.5, optimum pH 7.5-8.0 (8).
Related Products
- RNase A/T1 Mix
- Water, nuclease-free
Ebeling, W., et al., Proteinase K from Tritirachium album Limber, Eur. J. Biochem., 47, 91-97, 1974.
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.
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.
Brdiczka, D., Krebs, W., Localization of enzymes by means of proteases, Biochim. Biophys. Acta, 297, 203-212, 1973.
Crowe, J.S., et al., Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion, Nucleic Acids Res., 19, 184, 1991.
Jany, K.D., et al., Amino acid sequence of Proteinase K from mold Tritirachtum album Limber Proteinase K – a subtilisn related enzyme with disulfide bonds, FEBS Lett., 199, 139-144, 1986.
Bajorath, J, et al, The enzymatic activity of proteinase K is controled by calcium., Eur. J. Biochem., 176, 441-447, 1988.
Ardelt, W., Laskowski, M.Jr., Turkey ovomucoid third domain inhibits eight different serine proteinases of varied specificity on the same ...Leu18-Glu19... reactive site, Biochemistry, 24, 5313-5320, 1985.
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Updated balandžio 02, 2008 08:30
