How long does proteinase k last

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Local Sales Support. About Promega. Join Our Team. Contact Us. Your Cart. Current Items 0. Instructions for Use of Product s V Proteinase K, produced by the fungus Tritirachium album Limber, is a serine protease that exhibits a very broad cleavage specificity. When working with PBS, however, it can be a little tricky, which is possibly due to the pH still within optimal range, but on the lower end of that range.

Typically, adding proteinase K powder a little at a time while mixing into solution will help dissolve it into PBS. What proteinase K is known for is protecting nucleic acids from protein degradation. This occurs because proteinase K is capable of digesting proteins that would normally damage your sample.

This protocol further details how to use proteinase K to inactivate nucleases during your extraction procedures. The benefit of using proteinase K during DNA extraction is its ability to degrade a wide range of damaging nucleases.

However, if this question is specifically geared toward isolating DNA from other proteins, the phenol-chloroform extraction is another option useful for removing proteins from solution. However, this method is more toxic.

However the question absolutely deserves a defined space here. Proteinase K is a broad-spectrum serine protease within the subtilisin family of proteins. It got its name because of its originally discovered ability to hydrolize keratine. Breyer, J.

Schulz-Schaeffer, W. Veterinary Microbiology, , Charette, S. Ebeling, W. Proteinase K from Tritirachium album Limber. Biochem, 47 , Muller, A. Crystal structure of calcium-free proteinase K at 1. The Journal of Biological Chemistry, , Tullis, R.

Analytical Biochemistry, 1 , How do you inactivate proteinase K? What is the optimal temperature for proteinase K activation? What exactly is the relationship between proteinase K and calcium? What are the activators of proteinase K? How is proteinase K involved with cell lysis?

Why is knowing the activity of proteinase K important? What applications is proteinase K used for? How do you make proteinase K stock solution? Proteinase K is used for the destruction of proteins in cell lysates tissue, cell culture cells and for the release of nucleic acids, since it very effectively inactivates DNases and RNases.

Proteinase K is very useful in the isolation of highly native, undamaged DNAs or RNAs, since most microbial or mammalian DNases and RNases are rapidly inactivated by the enzyme, particularly in the presence of 0.

In contrast, when measured using peptide substrates, denaturants inhibit the enzyme. The reason for this result is that the denaturing agents unfold the protein substrates and make them more accessible to the protease. Proteinase K activity is greatly increased by the addition of denaturing agents like SDS or urea Hilz et al. The proteinase K seems to be a pretty stable enzyme, and can still work at this temperature. Activated by calcium 1 — 5 mM , the enzyme digests proteins preferentially after hydrophobic amino acids aliphatic, aromatic and other hydrophobic amino acids.

Although calcium ions do not affect the enzyme activity, they do contribute to its stability. Proteins will be completely digested, if the incubation time is long and the protease concentration high enough. Upon removal of the calcium ions, the stability of the enzyme is reduced, but the proteolytic activity remains. 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. Chromosomal DNA that has been embedded in agarose plugs can be treated with proteinase K to inactivate rare-cutting restriction enzymes used to digest the DNA. Isolation of plasmid and genomic DNA. Genomic or plasmid DNA can be isolated from liquid nitrogen frozen cells or cultured cells using proteinase K.