Proteinase K application technology
Protease k is a high activity protease of the subtilase class used for the general degradation of proteins in biological samples. Purified from Tritirachium album limber. The enzyme has two ca2+ binding sites, which are separated from the active center of the enzyme and are not directly related to the catalytic mechanism. However, if ca2+ is removed from the enzyme, the catalytic activity will be lost by about 80% due to remote structural changes, but the residual activity is usually sufficient to degrade the protein that normally contaminates the acid product. Therefore, edta is usually added during the digestion of protease k (to inhibit the action of nucleases dependent on mg2+). However, if a protein that is highly resistant to proteinase k, such as keratin, is to be digested, it may be necessary to use a buffer containing 1 mmol/l ca2+ without edta. After the digestion was completed, EGTA (ph8.0) was added to a final concentration of 2 mmol/l to purify the ca2+ before purifying the nucleic acid.
Proteinase K is a serine protease with a broad cleavage activity. It cleaves the carboxy terminal peptide bond of an aliphatic amino acid and an aromatic amino acid. This enzyme was purified to remove RNase and DNase activity. Since proteinase K is stable in urea and SDS and has the ability to degrade natural proteins, it is widely used, including preparation of chromosomal DNA for pulse electrophoresis, Western blotting, and removal of nucleases in DNA and RNA preparation. The working concentration of proteinase K is 50-100 μg/ml. It is active in a wide pH range (pH 4-12.5).
Recommended reaction buffer: 50 mM Tris-HCl (pH 7.5), 10 mM CaCl2.
It is worth noting that proteinase K is commonly used in in situ hybridization techniques for pre-hybridization treatments, which have the effect of digesting proteins surrounding the target DNA to increase the chance of binding of the probe to the target nucleic acid and increase the hybridization signal. If 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 destroyed, resulting in the shedding of the tissue sections and the disappearance of the nucleus, thus affecting the hybridization results. EDTA buffer can replace the action of proteinase K. Solve the above problems and achieve the desired dyeing effect.
Preservation: Store in a -20 °C refrigerator to avoid repeated freezing and thawing, effectively guarantee the December
Incubation temperature is 55 to 65 ° C, ideal incubation temperature is 58 ° C, incubation time is 15 minutes to 48 hours; ideal incubation time is 2 hours
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