Sample solution is provided at 25 µL, 10mM.
|Cas No.||39450-01-6||M.Wt||29.3 kDa|
|Specific activity||>30 U/mg||Stability||pH range:4.0 to 12.5, temperature range: 25°C to 65°C.|
|Solubility||Soluble in 20 mM Tris-HCl,1 mM CaCl2,50% Glycerol,pH 7.4||Storage||Store at -20°C|
|General tips||GlpBio guarantees optimal performance of this product for 18 months after date of delivery under the appropriate temperature and condition.|
|Shipping Condition||Evaluation sample solution : ship with blue ice|
Quality Control & SDS
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Specific activity: >30 U/mg
This protocol is used in the extraction of genomic DNA from mouse tail with Proteinase K.
1. Place an approximately 0.5cm mouse tail into a 1.5 ml microcentrifuge tube.
2. Add 500 μl of lysis buffer (For example: 50 mM KCl, 50 mM Tris-HCl (pH 8.0), 2.5 mM EDTA,0.45% NP-40, 0.45% Tween-20).
3. Add 2.5 μl Proteinase K (20 mg/ml) to the tubeand mixthe solution.
4. Incubate overnight at 55°C.
Optional step:Incubate for an additional 1 hour at 65°C.
5. Vortex the tube and spin down for 10 seconds at 13,000 rpm to collect cell debris.
6. Use 1 μl from the top part of the supernatant per 50 μl of PCR mix(for genotyping).
Proteinase K is a broad-spectrum serine protease and our product is extracted from Pichia pastoris cells with cloned gene encoding Engyodontium album (Tritirachium album) endolytic protease. It is a highly reactive protease frequently used for digesting various proteins and enzymes (including endonuclease, exonuclease, DNase or RNase) .Therefore, it is usually used in DNA preparations without impairing the integrity of the isolated DNA. It has a superior performance under a broad range of conditions: pH, buffer, detergents (such as SDS), chelator (such as EDTA), and temperature. Proteinase K hydrolyzes peptide bonds preferentially adjacent to carboxyl group of hydrophobic amino acids (aliphatic, aromatic, and others).
Except the isolation of genome, it can also take a job in detection of enzyme localization or removal of enzymes from DNA to improve cloning efficiency.
Appropriate working concentration of proteinase K is always among the range of 0.05 to 1 mg/mL. The activity of the enzyme can be stimulated by 0.2 to 1% SDS or by 1 to 4 mol urea. It is activated by calcium (1-5mM), although calcium ions do not affect the enzyme activity, but it contributes to the thermal stability and protects the proteinase from autolysis. Proteinase K has two binding sites for Ca2+, which are located close to the active center, but are not directly involved in the catalytic mechanism. So calcium ion has a regulatory function for the substrate binding site of proteinase K. The enzyme is inactivated by DIFP or PMSF. However, it is not inhibited by EDTA, iodoacetic acid, trypsin-specific inhibitor TLCK, chymotrypsin-specific inhibitor TPCK, and p-chloromercuribenzoate.
We recommend an optimum pH of 7.5 to 8.0 and optimum temperature at 50 to 55°C. Rapid denaturation will occur at temperatures above 65°C. You can hold it under 95°C for 10 min as a heat inactivation.
. Kraus, E; et.al. Proteinase K from the Mold Tritirachium album limber, Specificity and Mode of Action. Z. Physiol. Chem., 357:939; 1976.
. Jany,KD, et al. Amino Acid Sequence of Proteinase K from the Mold, Tritirachium albumlimber. Proteinase K; a Subtilisin-related Enzyme with Disulfide Bonds. FEBS Letter, 199,139.1986.