Proteinase K is a broad-spectrum serine protease (28.9kDa monomer) that cleaves peptide bonds at the carboxylic sides of aliphatic, aromatic, and hydrophobic amino acids.
Proteinase K is a broad-spectrum serine protease (28.9kDa monomer) that cleaves peptide bonds at the carboxylic sides of aliphatic, aromatic, and hydrophobic amino acids.
Applications
Isolation of genomic DNA from cultured cells and tissues, removal of DNases and RNases during DNA and/or RNA purification, determination of enzyme locations
Quantity
lyophilized powder purified from purified from Pichia pastoris harbouring the gene encoding endolytic protease from Tritirachium album.
Supplied with a 10x-concentrated Storage Buffer.
Specifications
Free of DNases and RNases. Specific Activity ~30 Kunitz units per mg.
Storage
Store lyophilized powder at +4ºC for several months or -20ºC for at least 2 years. The concentrated (10x) storage buffer (500mM Tris-HCl, 50mM CaCl2) can be stored at room temperature or +4ºC.
Enzyme activity
Proteinase K is activated by calcium (1-5mM). Although Ca2+ does not directly influences catalytic activity it does contributes to the protein stability (protection against autolysis, increasing thermal stability). Removal of Ca2+ (e.g. by adding EDTA) reduces proteolytic activity by 80%, however, the residual activity is sufficient to digest proteins, which usually contaminate nucleic acid preparations. Therefore, the digest with Proteinase K for the purification of nucleic acids is performed in the presence of EDTA (inhibition of magnesium-dependent enzymes such as DNases). Proteinase K is also stable over a wide pH range (4-12), with a pH optimum of
pH 8.0. Elevation of the reaction temperature from 37°C to 50 – 60°C may increase the activity several times, as might the addition of 0.5 – 1% SDS, 3M of Guanidinium chloride, 1Mof Guanidinium thiocyanate, and 4M urea. The recommended working concentration of Proteinase K is 0,05-1 mg/ml.
Inhibition
Proteinase K can be inhibited by phenylmethylsulfonyl fluoride (PMSF), trichloroacetic acid (TCA), 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), and diisopropyl phosphorofluoridate (DFP or DIFP). Proteinase K is NOT inhibited by metal chelators, thiol-reactive reagents, or by specific trypsin or chymotrypsin inhibitors (including SDS, Tween-20, Triton X-100, urea, EDTA, citrate, iodoacetic acid, Sarkosyl, Guanidinium chloride, Guanidinium thiocyanate, TLCK, and TPCK)
Inactivation
Proteinase K can be heat-inactivated at temperatures above 65°C
| GE010.0100 | Proteinase K (lyophilized, ~30KU/mg) with Buffer | 100mg |
| GE010.1000 | Proteinase K (lyophilized, ~30KU/mg) with Buffer | 1g |
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