Description
Proteinase K is a highly reactive nonspecific serine protease that belongs to the subtilisin family of proteins. It cleaves at the carboxylic acid side of aliphatic, aromatic, or hydrophobic amino acids. Proteinase K is capable of inactivating RNases and DNases and is used in the isolation or preparation of high molecular weight nucleic acids. Proteinase K is also useful for helping to characterize enzymes, due to its cleavage specificity. This enzyme was designated proteinase K because of its ability to hydrolyze keratin. Proteinase K is stable in a wide variety of detergents and buffer salts and at various temperatures and pH. The isoelectric point of proteinase K is 8.9.
Proteinase K is typically used at a concentration of 50-100 μg/ml. It is active with or without the presence of SDS, urea, EDTA or various metal ions, but the activity of proteinase K can be increased by adding the denaturing agents and the structure of proteinase K can be stabilized by addition of Ca 2+. Proteinase K is inactivated by heating to 95°C for 10 minutes or using an inhibitor such as PMSF, AEBSF or DFP.
GoldBio's Proteinase K undergoes two sterile filtration steps during the separation and purification processes. The final product is further analyzed by plate culture to ensure the removal of all living organisms was 100% effective.
Unit Definition: One unit is defined as the amount of enzyme that will liberate 1.0 μmol tyrosine (Folin-positive amino acid) from casein per minute at 37°C, pH 7.5.
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Applications
- Digestion of unwanted proteins
- Removal of endotoxins bound to cationic proteins such as lysozyme and RNaseA
- Removal of nucleases for in situ hybridization
- Prion research with respect to TSE (transmissible spongiform encephalopathies)
- Protease footprinting Mitochontrial isolation
- Isolation of genomic DNA
- Isolation of cytoplasmic RNA
- Isolation of highly native DNA or RNA
Storage/Handling
Store at -20°C.
Reconstitute in 50mM Tris-HCl (pH 8.0), 3mM CaCl2.
Proteinase K Quick Answers:
How do you inactivate proteinase K?
Inactivate proteinase K by heating it to 95˚C for 10 minutes. You can also permanently inactivate proteinase K by using protease inhibitors such as PMSF and AEBSF (Pefabloc®).
What is the best temperature for proteinase K activation?
The best temperature for proteinase K activity is between 50-65˚C. Higher temperatures help with protein unfolding, which makes it easier for proteinase K to break down proteins.
How do you store proteinase K?
Aliquot proteinase K stock solution and store at -20˚C for up to 1 year.
Store lyophilized proteinase K powder desiccated at -20˚C for up to 2 years.
How do you dissolve proteinase K?
Proteinase K is water soluble and it can also be dissolved in Tris or PBS. Dissolving proteinase K with PBS is a little challenging, which may be due to pH. It's best to add proteinase K a little bit at a time while mixing into solution.
What is the optimal pH when working with proteinase K?
The optimal pH when working with proteinase K is between 7.5 - 12.0
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