Proteinase K (lyophilized) with Buffer 

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.

SKU:N/A

Category:Enzymes

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

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