Q : Why do we neutralize the proteinase K after incubation ? R : The protein concentration of the enzyme is ~20 mg/ml and its unit activity is 800 U/ml. Q : What is the protein concentration of Proteinase K, Molecular Biology Grade ? Proteinase K is used in many DNA extraction protocols to digest these contaminating proteins.Īdditionally, nucleases (enzymes that break down nucleic acids) may be present, the addition of proteinase K degrades these nucleases and protects the nucleic acids from nuclease attack. ◉ What is the function of proteinase k in DNA extraction?ĭuring the extraction of DNA (or nucleic acids in general), many contaminating proteins are present. Small amount of activity remaining through this method.), trichloroacetic acid (TCA) or serine protease inhibitors AEBSF, PMSF or DFP (permanently inactivate proteinase K) Proteinase K is inactivated by heating at 95☌ for 10 minutes ( heating does not fully inactivate the enzyme, there will always be a
This enzyme is very useful in the isolation of highly native and undamaged DNA or RNA, since most microbial or mammalian DNases and RNases are rapidly inactivated by the enzyme, especially in the presence of 0.5 at 1% SDS. ◉ It is also useful for characterizing proteins : Due to the cleavage specificity of proteinase K, characteristic fragments of proteins are obtained which are useful for determining the structure and function of proteins. ◉ It is used for the destruction of proteins in cell lysates (tissues, cell culture cells) and for the release of nucleic acids Proteinase k is suitable for this application because the enzyme is active in the presence of protein denaturing chemicals such as SDS and urea, chelating agents such as EDTA, hydrogen sulfide reagents and trypsin or chymotrypsin inhibitors. That would otherwise degrade a desired DNA or RNA sample. ◉ Proteinase K is commonly used in molecular biology to digest proteins, remove contaminations, and inactivate DNases and RNases Short term storage at ambient temperatures do not harm the enzyme’s activity and stability. It has a shelf life of 12 months when stored in a dry place at 4–8☌, due to the fact that it is a very stable. Increasing the reaction temperature from 37☌ to 50-60☌ can increase activity many-fold, by making its substrate cleavage sites more accessible, as can adding 0.5-1% of sodium dodecyl sulfate (SDS) or guanidinium chloride (3 M), guanidinium thiocyanate (1 M) and urea (4 M). Proteinase K is stable over a wide range of pH (from 4 to 12) and temperature (from +37☌ to +65☌). RNase will break down contaminating RNA and Proteinase K will break down damaged proteins, DNases and RNases. Proteinase K and RNases are usually added together in lysis buffer because they form an efficient combination. EDTA does not directly affect enzyme activity, it can impact the calcium and therefore reduce proteinase K activity to some extent. (necessary to inhibit metal ion-dependent enzymes, such as nucleases). Proteinase K digestion for nucleic acid purification is usually performed in the presence of EDTA by using EDTA), the stability of the enzyme is reduced, but the proteolytic activity is maintained. Its stability (thermostability and protects proteinase K from autolysis), if calcium ions are removed (e.g. The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha-amino groups.Ĭalcium ion is the cofactor of proteinase K enzyme, calcium ions do not affect the activity of the enzyme but contribute to This enzyme belongs to the family of S8 peptidases. (there are two types of serine proteases, chymotrypsin and subtilisin). Proteases are enzymes that catalyze the cleavage of a protein into amino acids, proteinase K is a broad-spectrum serine protease belonging to the subtilisin class Proteinase K = Protein (long chain of amino acids) + ase (suffix for enzyme) + K (keratin)
Proteinase K CAS number 3 Molecular mass 391.466 g/Mol Formula C 29H 27N 2O 12P Synonyms Endopeptidase K,
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