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Proteinase K from Tritirachium album belongs to the familiy of subtilisin-like serine proteases. It has an endo- and exoproteolytic activity. Activated by calcium (1 - 5mM), Proteinase K exhibits a broad substrate specificity. It degrades many proteins in the native state even in the presence of detergents after hydrophobic amino acids. Proteins will be completely digested, if the incubation time is long and the protease concentration high enough.
As Calcium is not directly involved in the catalytic process, removal of the Calcium ions will almost not influence the enzymatic activity but the stability of the enzyme is reduced (about 80% residual activity). The pH-optimum is at 8, but the enzyme is active over a wide pH-range (pH5 - 12). An elevation of the reaction temperature from 37°C to 50 - 60°C may increase the activity several times, like the addition of 0.5 - 1% SDS. Temperatures above 65°C, trichloroacetic acid or the serine protease inhibitors AEBSF (M6360) >, PMSF (M3194) > or DFP inhibit the activity. For complete denaturation temperatures above 95°C are necessary.
Proteinase K will not be inhibited by EDTA, urea (1 - 4 M), SDS, citrate, iodoacetic acid or, interestingly, by other serine protease inhibitors like TLCK and TPCK. In case that proteinase K has to be inactivated, make sure, that the temperature is not below 95°C and the time not shorter than 10 minutes. A TCA-precipitation is well suited, too.
Proteinase K is used for the digestion of proteins in cell lysates (tissue, cell culture cells) and for the release of nucleic acids, since it very effectively inactivates DNases and RNases. The digest with Proteinase K for the purification of nucleic acids is performed in the presence of EDTA (inhibition of magnesium-dependent enzymes).
Conc. 20mg/mL proteinase K
Activity: min. 600 mAnsonU/mL (>600 units/mL)
spec. Activity: >30 mAnsonU/mg (>30 units/mg)
application:Protein digest in DNA samples for example: Purification of genomic DNA from bacteria (miniprep): Bacteria from a saturated liquid culture are lysed and proteins are removed by a digest with 100μg/mL Proteinase K for 1 h at 37°C. Whole-Mount in situ hybridization and determination of RNAs in vertebrate embryos and isolated organs: Digest of the sample with e. g. 10μg/mL Proteinase K for 15 minutes at room temperature. The period of the treatment and/or the concentration of the enzyme has to be optimized. Prepration of DNA from cells or tissue for PCR: Cells or tissue are incubated over night at 50°C with 100μg/mL Proteinase K. Isolation of vaccinia virus DNA: Digest the virus in a suspension with 2mg/mL Proteinase K for 4 h at 37°C.
Unit Definition:Concentration: 20mg/mL, Activity: >30 U/mg (>600U/mL). 1 unit is definded as the enzyme acitivity which liberates folin-positive amino acids and peptides corresponding to 1 µmol tyrosine in 1 minute at 37°C using haemoglobin as substrate.
Sicherheits Hinweise / SafetyH-Sätze: H315, H319, H335
GHS-Symbole: GHS07, GHS08
Klassifizierungen / ClassificationEC-Nr: 254-457-8
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