85-5965-33　［取扱停止］Proteinase K, NGS Grade, ≥40 Units/mg 500mg　CAS No:39450-01-6　P9100-01

特徴

• Proteinase K from Parengyodontium album (Tritirachium album) is a subtilisin-related serine protease. It is a broad spectrum endopeptidase with very high specific activity. It is widely used for digestion of proteins, including DNases and RNases, during nucleic acid preparations without compromising integrity of isolated DNA or RNA. Proteinase K is active under wide range of reaction conditions, including elevated temperatures and presence of SDS. This recombinant enzyme is expressed in Pichia pastoris, and undergoes extensive purification to yield the highest quality product. An extra purification step results in significantly increased solubility (2.5 fold), increased specific activity, and decreased DNA content, compared to our Molecular Biology Grade product.
• 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 7.5-8.02. Not inactivated by metal chelators, by thiol-reactive reagents or by specific trypsin and chymotrypsin inhibitors3. Activity of the enzyme is stimulated by 0.2-1% SDS or by 1-4M urea4. 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 proteins2. Isolation of genomic DNA and mRNA from cultured cells3. Removal of DNases and RNases when isolating DNA and RNA from tissues or cell lines4. Determination of enzyme localization5. Improving cloning efficiency of PCR products
• Synonyms:EC=3.4.21.64; Tritirachium album limber; Endopeptidase K; PROK; Tritirachium Alkaline Proteinase
• Molecular Weight:28.9kD
• Form:Supplied as a lyophilized powder.
• Source:Tritirachium album limber
• Specific Activity (Protein):≥35 units/mg dry weight
• Unit Definition:One unit liberates 1umole of Folin positive amino acids, measured as Tyrosine, in 1 minute at 37°C, pH 7.5, using urea-denatured hemoglobin as the substrate.
• DNA Content:≤5pg/mg
• Working Concentration:3ul of a 20mg/ml solution per 1.5ml of bacterial culture. Dissolve 20mg/ml Proteinase K in 10mM Tris HCl, pH 7.5, 1mM calcium acetate.
• 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.
• Recommended Storage Buffer:50mM Tris-HCl, pH 8, 5mM CaCl2 and 50% (v/v) glycerol (storage at -20ºC)
• Quality Control:Exonucleases: None DetectedEndonucleases: None DetectedRibonucleases: None Detected
• Storage and Stability:Lyophilized powder may be stored at 4°C. Stable for 24 months after receipt at 4°C. Reconstitute, aliquot and store at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
• 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.