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Tritirachium album proteinase K
Proteinase K is a serine protease with wide cleavage activity. It is also stable in the presence of SDS, urea or EDTA. Its relative molecular weight is about 29.3 kDa, which can cleave the carboxy-terminal peptide bonds of aliphatic and aromatic amino acids and is used for protein degradation in biological samples.
Proteinase K is a stable S8 family serine alkaline protease, which contains two disulfide bonds and one free cysteine adjacent to the active site histidine.
28,930 Da (amino acid sequence)
28,500 Da (SDS-PAGE)
pH range: 7.5-12.0 (heme after urea denaturation is the substrate), but the pH range used in most cases is 7.5-9.0.
Temperature curve: the optimum activity temperature is 37℃ (the activity between 20 and 60℃> 80% of the maximum activity)
Extinction coefficient: E1% = 14.2 (280 nm, 10 mM NaCl and 5 mM CaCl2, pH 8.0)
Activator: 1-5 mM Ca2+ is required for activation. After removing the calcium ions in the enzyme (adding EDTA), 25% of the catalytic activity will be lost. However, if the EDTA-Ca2+ complex is removed by gel filtration, a total of 80% of the enzyme activity will be lost, and only a small amount of activation will occur when excess Ca2+ is added to the enzyme without Ca2+.
Proteinase K is activated in 1% TRITON™ X-100 and fully activated in 0.5% (w/v) SDS. SDS and urea will denature protein substrates and increase the rate of digestion. Under the action of these reagents, the digestion rate of proteinase K itself is much slower.
Unit definition: At pH 7.5 and 37℃, the amount of enzyme that can hydrolyze urea-denatured heme per minute to produce 1.0 mmole (181 mg) of tyrosine is one unit.
Dilution buffer: 20mM Tris-HCl (pH7.4), 1mM CaCl2
Storage buffer: 20mM Tris-HCl (pH7.4), 1mM CaCl2, 50% Glycerol
Proteinase K was found in the extract of Engyodontium album in 1974. It can digest natural keratin, so it is named proteinase K.
20mg/ml proteinase K preparation method
Add 200 mg of proteinase K to 9.5 ml of water and shake gently until proteinase K is completely dissolved. Do not vortex to mix. Add water to make the volume up to 10ml, then divide into small portions and store at -20℃.
Proteinase K solution stability
Proteinase K can be active in a wide range of pH 4-12(Best for pH8.0) and high temperature(30~70℃). At pH 8.0, the proteinase K solution can be stored stably for at least 12 months at 4 ℃. At pH 4-11.5, the proteinase K solution containing Ca2+ (1-6 mM) is expected to be stable for several weeks. 80% ammonium sulfate suspension can be stored stable for at least 12 months at 4 ℃.
The inhibitor of proteinase K is DIFP or PMSF (the latter uses a final concentration of 5 mM). EDTA will only partially inactivate it, but it cannot inhibit it. Proteinase K is not inhibited by iodoacetic acid, trypsin-specific inhibitor TLCK, chymotrypsin-specific inhibitor TPCK, and p-chloromercuryl benzoate.
In the extraction of DNA/RNA, the main function of proteinase K is to enzymolyze the histone bound to the nucleic acid to free the DNA/RNA in the solution, and then use different methods for extraction to remove impurities and collect the DNA/RNA.
1. Mitochondrial separation
2. Protein digestion of nucleic acid purification products. In molecular biology applications, proteinase K is often used to digest useless proteins, such as digestion of nucleases from DNA or RNA preparations of microorganisms, cultured cells, and plants. In nucleic acid preparations, the concentration of this enzyme is usually 50-200 µg/ml, pH 7.5-8.0, 37℃. The incubation time varies from 30 minutes to 18 hours. Although proteinase K can be automatically digested during long-term incubation, it is usually denatured by subsequent partial extraction methods.
3. Proteinase K has been used to remove endotoxins bound to cationic proteins, such as lysozyme and ribonuclease A.
4. Determine the distribution of enzymes on the membrane
5. Treat paraffin-embedded tissues to expose antigen binding sites for antibody labeling
6. Nuclease removal for in situ hybridization
7. Infectious spongiform encephalopathy (TSE) prion research and draft diagnostic test methods use proteinase K to digest proteins from brain tissue samples.
8. The proteinase K digestion method was used for the protease footprint experiment to remove protein-protein surface interaction.
The proteinase K gene comes from the tritirachium album limber and is obtained through yeast recombinant expression, reaching the molecular biology level. Proteinase K has a wide range of applications. It is mainly used to remove nucleases in DNA and RNA preparation. It can also be used to degrade protein impurities in non-protein component preparation systems, such as DNA vaccine preparation and heparin preparation. Proteinase K can also be used to prepare chromosomal DNA for pulse electrophoresis and Western blot experiments. Denaturants such as SDS (1%) can increase its activity. The usual working concentration of proteinase K is 50-100 µg/ml. The specific working concentration is determined according to whether the buffer used contains SDS, urea, pH, temperature and other factors.
Precautions for use
For your safety and health, please wear lab coats and disposable gloves.
If you need to use it repeatedly after activation, you need to freeze it in the refrigerator at -20℃～-80℃ or make it into a solution and filter, sterilize, and store it separately.
The concentration of proteinase K solution is generally 10 mg/ml or 20 mg/ml. The proteinase K solution is colorless and transparent. If precipitation occurs, it cannot be used.
100mg/bottle or as your requirements
Storage and transportation methods
Store at -20℃ in powder form, avoid repeated freezing and thawing. It can be stable for at least 2 years. Use a sealed foam box with built-in ice pack for transportation.