Reagents and instruments for immunology, cell biology and molecular biology.
 
> Proteinase K, ≥30 Units/mg

Proteinase K, ≥30 Units/mg

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Cat# P9100-100mg

Size : 100mg

Brand : US Biological

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Phone : +1 850 650 7790

P9100 Rabbit Anti-Proteinase K, ≥30 Units/mg

Clone Type
Polyclonal
Grade
Molecular Biology Grade
Shipping Temp
RT
Storage Temp
-20°C

A non-specific serine protease, Proteinase K will inactivate nucleases during native mRNA and DNA preparation. 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. Recommended for high molecular weight nucleic acids from mammalian and microorganism sources. Used in the preparation of genomic DNA from bacteria. Active in the presence of SDS.||Properties:|1. Stable over a wide pH range: 4.0-12.5, optimum pH 7.5-8.0|2. Not inactivated by metal chelators, by thiol-reactive reagents or by specific trypsin and chymotrypsin inhibitors|3. Activity of the enzyme is stimulated by 0.2-1% SDS or by 1-4M urea|4. Ca2+ protects Proteinase K against autolysis, increases the thermal stability and has a regulatory function for the substrate binding site of Proteinase K||Applications:|1. Purification of target material from contaminating proteins|2. 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|4. Determination of enzyme localization|5. Improving cloning efficiency of PCR products| |Synonyms:|EC=3.4.21.64; Tritirachium album limber; Endopeptidase K; PROK; Tritirachium Alkaline Proteinase||CAS No:|39450-01-6||Molecular Weight:|28.9kD||Appearance:|Supplied as a lyophilized powder.||Specific Activity (Protein): |≥30 units/mg dry weight||Recommended Preparation:|Dissolve 20mg/ml Proteinase K in 50mM Tris-HCl, pH 7.8, 3mM calcium acetate for immediate use or 50mM Tris-HCl, pH 7.8, 3mM calcium acetate, 50% glycerol for long term storage at -20ºC.||Unit Definition:|One unit of proteinase K hydrolyzes urea-denatured hemoglobin producing color equivalent of 1umol tyrosine per minute at 37ºC, pH 7.5 (Folin & Ciocalteu’s method), 1U=1mAnsonU.||Working Concentration:|3ul of a 20mg/ml solution per 1.5ml of bacterial culture.||Optimum pH:|7.5-12, using denatured hemoglobin as substrate.||Inhibitors:|Phenylmethylsulfonyl fluoride (PMSF) and diisopropyl phosphorofluoridate (DPF) completely inhibit the enzyme. Proteinase K is also inactivated by heating above 65ºC for 20 min.||Specificity:|In addition to cleavage of peptide bonds, it is able to catalyze peptide amide hydrolysis. Proteinase K is inactivated by diisopropyl fluorophosphate (DFP) or phenyl methane sulphonyl fluoride (PMSF). Chelating agents such as citrate and EDTA have no affect on the enzyme activity.||Quality Control:|The product passed the quality tests for the presence of endo- and exonucleases ||Storage and Stability:|Lyophilized and reconstituted products are stable for 6 months after receipt at -20°C. Reconstitute with sterile recommended storage buffer (above). Aliquot to avoid repeated freezing and thawing. Store at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. ||Important Note:|Proteinase K solution contains Ca2+ and glycerol and is stable for 6 months. Shipping and short term storage can be at ambient temperature. Although calcium ions do not affect the enzyme activity, they do contribute to its stability when present at a concentration of 1-5 umoles. An interesting characteristic of proteinase K is that it retains its activity in the presence of SDS or urea. (0.5-1% SDS and 1-4 M urea). Raising the temperature of the reaction from 37°C to 50-60°C can increase the activity several fold. A special feature of proteinase K is its ability to digest native proteins, thereby inactivating enzymes such as DNase and RNase without recourse to a denaturation process.

Applications
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. ||Toxicity and Hazards: All products should be handled by qualified personnel only, trained in laboratory procedures.
References
1. Ellwood, S.R., et al., Genome Biology 11: R109 (2010). General References: 1. Ebeling, W., et al., Eur. J. Biochem, 47: 91-97 (1974). 2. Ausubel, F.M., et al., “Current Protocols in Molecular Biology”, John Wiley (1992). 3. Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). 4. Bernabeu, C., et al., Eur. J. Biochem., 93, 527 (1979). 5. Betzel, C., Pal, G., Struck, M., Jany, K., and Saenger, W.: Active-Site Geometry of Proteinase K: Crystallographic Study of Its Complex with a Dipeptide Chloromethyl Ketone Inhibitor, FEBS Lett., 197, 105 (1986). 6. Brdicka, D., and Krebs, W.: Localization of Enzymes by Means of Proteases, Biochim. Biophys. Acta, 297, 203 (1972). 7. Broemme, D., Peters, K., Fink, S., and Fittkau, S.: Enzyme-Substrate Interactions in the Hydrolysis of Peptide Substrates by Thermitase, Subtilisin BPN’, and Proteinase K, Arch. Biochem. Biophys., 244, 439 (1986).Crowe, J., Cooper, H., Smith, M., Sims, M., Parker, D., and Gewert, D.: Improved Cloning Efficiency of Polymerase Chain Reaction (PCR) Products After Proteinase-K Digestion, Nucleic Acids Res., 19, 184 (1991).Cryer, D., Eccleshall, R., and Marmur, J.: Isolation of Yeast DNA in Methods in Cell Biology, Vol XII, (Prescott, D., ed, Academic Press, NY), 39 (1975).Dattagupta, J., Fujiwara, T., Grishin, E., Lindner, K., Manor, P., Pieniazek, N., Saenger, W., and Suck, D.: Crystallization of the Fungal Enzyme Proteinase K and Amino Acid Composition, J. Mol. Biol., 97, 267 (1975).Ebeling, W., Hennrich, N., Klockow, M., Metz, H., Orth, H., and Lang, F.: Proteinase K from Tritirachium album Limber, Eur. J. Biochem., 47, 91 (1974).Fourcroy, P.: Isolation of Undegraded Polysomes from Radish Cotyledons: Use of Proteinase K and Cycloheximide, Phytochemistry, 19, 7 (1980).Gajda, A., Rudenskaya, G., and Stepanov, V.: Isolation and Comparative Properties of Serine Proteinases of the Microscopic Fungi Trichoderma lignorum and Trichoderma koningii, Biokhimiya, 46, 2064 (1981).Hansen, J.: Isolation of Higher Molecular Weight DNA from Bacillus cereus T Using Proteinase K, Prep. Biochem., 4, 473 (1974).Hilz, H., Wiegers, U., and Adamietz, P.: 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 (1975).Holm, C., Meeks-Wagner, D., Fangman, W., and Botstein, D.: A Rapid, Efficient Method for Isolating DNA from Yeast, Gene, 42, 169 (1986).Jany, K., Lederer, G., and Mayer, B.: Amino Acid Sequence of Proteinase K from the Mould of Tritirachium album Limber, FEBS Lett, 199, 139 (1986).any, K., and Mayer, B.: Proteinase K from Tritirachium Album limber: I. Molecular Mass and Sequence around the Active Site Serine Residue, Biol. Chem. Hoppe-Seyler, 366, 485 (1985).||Jany, K., Lederer, G., and Mayer, B.: Amino Acid Sequence of Proteinase K from the Mold Tritirachium Album limber. Proteinase K: A Subtilisin-related Enzyme with Disulfide Bonds, FEBS Lett., 199, 139 (1986).||Jehanli, A., and Hough, D.: Pronase and Proteinase K Digestion of Human Immunoglobulin M, Mol. Immunol., 22, 557 (1985).||Kasche, V., Zollner, R., Amneus, H., and Naslund, L.: A Two-Step Procedure for Quantitative Isolation of Pure Double Strand DNA From Animal Tissus and Cell Cultures, Prep. Biochem., 11, 233 (1981).||Kraus, E., and Femfert, U.: Proteinase K from the Mold Tritarachium album Limber, Specificity and Mode of Action, Z. Physiol. Chem., 357, 937 (1976).||Lizardi, P., and Engelberg, A.: Rapid Isolation of RNA Using Proteinase K and Sodium Perchlorate, Anal. Biochem., 98, 116 (1979).||Lough, J., Wrenn, D., Miziorko, H., and Auer, H.: Differential Sensitivity of Chicken MM-Creatine Kinase to Trypsin and Proteinase K, Intl. J. Biochem., 17, 309 (1985).||McCormick, J., Larson, L., and Maher, V.: Problems in the Extraction of DNA when Utilizing Pancreatic RNAase and Pronase, Biochim. Biophys. Acta, 349, 145 (1974).||McGodkin, R.: RNA Extraction by the Proteinase K Method, Methods Mol. Biol., 2, 109 (1984).||Mendolsohn, S., and Young, D.: Inhibition of Ribonuclease: Efficacy of Sodium Dodecyl Sulfate, Diethyl Pyrocarbonate, Proteinase K and Heparin Using a Sensitive Ribonuclease Assay, Biochim. Biophys. Acta, 519, 461 (1978).||Morihara, K., and Tsuzuki, H.: Specificity of Proteinase K from Tritirachium album Limber for Synthetic Peptides, Agr. Biol. Chem., 39, 1489 (1975).||Orstan, A., and Gafni, A.: Inhibition of Proteinase-K by Phosphorylated Sugars, Biochem. Intl., 25, 657 (1991).||Paehler, A., Banerjee, A., Dattagupta, J., Fujiwara, T., Lindner, K., Pal, G., Suck, D., Weber, G., and Saenger, W.: Three-Dimensional Structure of Fungal Proteinase K Reveals Similarity to Bacterial Subtilisin, EMBO Jrnl., 3, 1311 (1984).||Pal, G., Betzel, C., Jany, K., and Saenger, W.: Crystallization of the Bifunctional Proteinase/Amylase Inhibitor PKI-3 and of Its Complex with Proteinase K, FEBS Lett., 197, 111 (1986).||Prescott, D., ed.: Isolation of Yeast DNA, in Methods in Biol., Vol. XII, 39 (1975).||Rauber, N., Jany, K., and Pfleiderer, G.: RNase A Digestion by Proteinase K , Z. Naturforsch. Sect. C, 33, 9 (1978).||Roelcke, D., and Uhlenbruck, G.: Proteinase K: A New Serological Effective Protease from Fungi, Z. Med. Mikrobiol. Imm., 155, 156 (1969).||Veerisetty, V., and Sehgal, O.: Proteinase K–Sensitive Factor Essential for the Infectivity of Southern Bean Mosaic Virus Ribonucleic Acid, Phytopathology, 70, 282 (1980).||Vidales, F., Bernabeu, C., and Ballesta, J.: Peptidyl Transfer Ribonucleic Acid Hydrolase Activity of Proteinase K, Biochemistry, 18, 4155 (1979).||Wiegers, U., and Hilz, H.: A New Method Using Proteinase K to Prevent mRNA Degradation During Isolation from HeLa Cells, Biochem. Biophys. Res. Comm., 44, 513 (1971).||Wiegers, U., and Hilz, H.: Rapid Isolation of Undegraded Polysomal RNA without Phenol, FEBS Lett., 23, 77 (1972).