Description

Background

What is proteinase K used for? Proteinase K is commonly used for protein digestion, DNA extraction, RNA purification, direct PCR, and genotyping. Since it very effectively inactivates DNases and RNases, the recombinant proteinase K enzyme is added during nucleic acid preparations to isolate highly native, undamaged DNAs or RNAs; it is used in direct PCR kits for genotyping by destruction of proteins in cell lysates (tissue or cell culture cells) and for release of genomic DNA.

What is the function of proteinase K? Proteinase K is a broad-spectrum serine protease originally isolated from fungus Engyodontium album. The protease was named “Proteinase K” for its ability to digest Keratin. Crystal and molecular structure studies suggest that the enzyme belongs to the subtilisin family characterized with a catalytic triad (Asp39-His69-Ser224) in active site. The proteinase K enzyme has no pronounced cleavage specificity and preferential cleavage site is the peptide bond adjacent hydrophobic amino acids.

The proteinase K enzyme exhibits higher proteolytic activity in the presence of reducing agents such as 5 mM DTT. Proteinase K is inhibited by serine protease inhibitors such as PMSF, DFP, and AEBSF.

Proteinase K is active in 1% Triton X-100 and fully active in 0.5% (w/v) SDS which denatures protein substrates to increase digestion rates. The enzyme works best at 50-200 ug/mL at pH 7.5-8.0, 37 °C and is usually denatured by subsequent phenol extractions. Incubation times vary from 30 minutes to 18 hours and proteinase K can auto-digest during long incubation.

PCR enzymes:

* Taq DNA polymerase.
* High yield Taq DNA polymerase.
* Bst DNA polymerase large fragment.
* Hot-start high-fidelity DNA polymerase.
* Hot start Taq DNA polymerase with chemical or antibody inhibitor, used for SYBR green or Taqman probe-based real time PCR (qPCR or quantitative PCR).

2x PCR master mixes:
* 2x PCR preMix with dye (green, red, blue).
* 2x rapid PCR master mix with a green dye.
* 2x long PCR master mix with a blue dye.
* 2x high yield PCR master mix with a blue dye.
* 2x hot-start high-fidelity PCR master mix with a blue dye.
* Blood direct PCR kit
* Cell and tissue direct PCR kit
* RT supermix for qPCR.
* RT-PCR Kit.
* Dye-based one-step qRT-PCR kits.
* Probe-based one-step qRT-PCR kits.
* Probe-based multiplex one-step qRT-PCR kits.
* SYBR Green qPCR master mixes.
* Probe qPCR master mixes.

PCR components and related products:
* dNTP mix, 10 mM each and dNTP mix, 25 mM each.
* Proteinase K powder and Proteinase K solution.
* Reverse transcriptases and First strand cDNA synthesis kits.
* Murine RNase inhibitor.
* Uracil DNA glycosylases.
* PCR enhancer.

Questions and Answers:

Question: Can the proteinase K enzyme be used for direct PCR?
Answer: Yes. The enzyme can be used for isolation of genomic DNA from mouse tail and cultured cells, and removal of DNases and RNases when isolating DNA and RNA from tissues or cell lines. A simple "Universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification.

Question: Can you provide any proteinase K buffer recipe?
Answer: Depending on the specific applications of the enzyme, the proteinase K buffer recipe may be different. Our recombinant proteinase K powder can be dissolved in water or storage buffer: 20 mM Tris-HCl, 1 mM CaCl2, 50% Glycerol, pH 7.4.

Question: Does the recombinant proteinase K enzyme work at room temperature?
Answer: The recommended temperature range for proteinase K digestion is 37-65°C; optimum activity at 50 to 55°C; maximum activity at 70°C and rapid denaturation of enzyme occurs at temperatures above 65°C.

Question: How to make Proteinase K solution from powder?
Answer: The most common proteinase K stock solution of 20 mg/ml is prepared by dissolving the lyophilized proteinase K powder in sterile water (stable for 1 year at −20°C) or in a solution of 20 mM Tris-HCl, 1 mM CaCl2, 50% Glycerol, pH 7.4 (stable for months at 4°C).

Question: How to prepare proteinase K work solution?
Answer: Proteinase K is generally used at a working concentration of up to 50 ug/ml in various buffer formulations, including those that contain up to 0.5% SDS.

Question: How to inactivate the recombinant proteinase K?
Answer: The most recommended proteinase K inactivation method is to heat the recombinant proteinase K enzyme to 95°C for 10 minutes. However, the enzyme cannot be completely inactivated by heating. The subsequent washing is also recommended to remove the leftover enzyme.

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Proteinase K

Peptidase K, Endoproteinase K, Endopeptidase K, Protease K, Endopeptidase K, Tritirachium Alkaline Proteinase, Tritirachium Album Serine Proteinase

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