Introduction to Proteinase K

Proteinase K is a powerful proteolytic enzyme isolated from Candida albicans, which has high specific activity and is a key reagent for DNA extraction. The enzyme is active in a wide pH range (4~12.5) and high temperature (50~70°C), and is used for the separation of plasmid or genomic DNA and RNA. In DNA extraction, the main function is to enzymatically hydrolyze histones bound to nucleic acid, so that DNA is free in the solution, and then extracted by different methods to remove impurities and collect DNA. Chelating agents such as EDTA or detergents such as SDS cannot inactivate it. Proteinase K stock solution is generally 10mg/ml or 20mg/ml. Store at -20°C. Proteinase K solution is colorless and transparent, if precipitation occurs, it can no longer be used.

Introduction

Purified from Tritirachium album limber. According to the data, the enzyme has two Ca2+ binding sites, which are a certain distance away from the active center of the enzyme, and have no direct relationship with the catalytic mechanism. However, if Ca2+ is removed from the enzyme, about 80% of the catalytic activity is lost due to long-range structural changes, but the remaining activity is usually sufficient to degrade proteins that normally contaminate acid preparations. Therefore, EDTA is usually added during proteinase K digestion (to inhibit the action of Mg2+-dependent nucleases). However, if you want to digest proteins with strong resistance to proteinase k, such as keratin, you may need to use a buffer containing 1mmol/l Ca2+ without EDTA. After digestion and before purification of nucleic acid, EGTA (ph8.0) should be added to a final concentration of 2mmol/l to chelate Ca2+.

Proteinase K is a serine protease with broad cleavage activity. It cleaves the carboxy-terminal peptide bonds of aliphatic and aromatic amino acids. The enzyme is purified to remove RNase and DNase activity. Because proteinase K is stable in urea and SDS and has the ability to degrade native proteins, it has a wide range of applications, including the preparation of chromosomal DNA for pulse electrophoresis, western blotting, and the removal of nucleases from DNA and RNA preparations. The general working concentration of proteinase K is 50-100μg/ml. It is active over a wide pH range (pH 4-12.5).

Recommended reaction buffer: 50mM Tris-HCl (pH7.5), 10mM CaCl2.

Proteinase K preparation standard: 20mg/ml proteinase K preparation standard (proteinase K): Add 200mg of proteinase K to 9.5ml of water, shake gently until proteinase K is completely dissolved. Do not vortex to mix. Add water to make up to 10ml, then aliquot into small portions and store at -20°C.

Inhibitor

Proteinase K is a serine protease, so its action can be inhibited with phenylmethylsulfonyl fluoride, an inhibitor of serine proteases.

Effect

It is worth noting that proteinase K is usually used for pre-hybridization treatment in in situ hybridization technology. It has the function of digesting the protein surrounding the target DNA to increase the chance of the probe binding to the target nucleic acid and improve the hybridization signal. However, proteinase K When the concentration is too high, the digestion time is too long or the incubation temperature is too high, the structure of the cells will be damaged to a certain extent, resulting in the detachment of the tissue slices and the disappearance of the nucleus, thus affecting the hybridization results. EDTA buffer can replace the role of proteinase K, Solve the above problems, and can achieve the ideal dyeing effect.

Preservation method

Preservation: Store in -20°C refrigerator, avoid repeated freezing and thawing, effective guarantee for 12 months.

The incubation temperature is 55 to 65°C, the ideal incubation temperature is 58°C, and the incubation time is 15 minutes to 48 hours; the ideal incubation time is 2 hours.

Application

Proteinase K has been widely used in leather, fur, silk, medicine, food, brewing and so on. The depilation and softening of the leather industry has made extensive use of protease, which not only saves time, but also improves labor hygiene conditions. Protease can also be used for silk degumming, meat tenderization, wine clarification. Clinically, it can be used as medicine, such as treating dyspepsia with pepsin, treating bronchitis with acid protease, treating vasculitis with elastase, and using trypsin and chymotrypsin to purify surgical suppurative wounds and interthoracic serosa Adhesion treatment. Enzyme laundry detergent is a new product in detergents. It contains alkaline protease, which can remove blood stains and protein stains on clothes, but be careful not to touch the skin when using it, so as not to damage the protein on the skin surface and cause allergic phenomena such as rashes and eczema.