Proteinase K is a subtilisin-related serine protease that has no pronounced cleavage specificity.
The enzyme is also available as a lyophilizate. The recombinant enzyme is identical to the native protease originally isolated from the mold Tritirachium album. The specifications of the recombinant enzyme are the same as those of the native protease. The amino acid sequence (molecular weight) and the molecule structure are identical. However, the recombinant preparation is much purer than the native enzyme, as it is DNA-free, according to the current quality control procedures, and is therefore well suited for isolating PCR and RT-PCR templates.
- This PCR grade Proteinase K is extremely effective on native proteins and can therefore be used to rapidly inactivate endogenous nucleases such as RNases and DNases. This property makes Proteinase K particularly suitable for the isolation of native RNA and DNA from tissues or cell lines.
- The enzyme promotes cell lysis by activating a bacterial autolytic factor.
- Proteinase K is also used for the analysis of membrane structures by modifying proteins and glycoproteins on cell surfaces.
- The enzyme is particularly well suited for isolating nucleic acids for amplification reactions.
- Proteinase K can be used to remove cellular debris during the preparation of colony lifts, and to treat tissue sections to ensure efficient probe infiltration during in situ hybridization.
Features and Benefits
Proteinase K cleaves proteins between amino acids X and Y (X-↓-Y), when X = an aliphatic, aromatic, or hydrophobic amino acid, and Y = any amino acid. The enzyme is extremely effective on native proteins and can therefore be used to rapidly inactivate endogenous nucleases such as RNases and DNases.
- Choose an effective tool for template preparation. Inactivate DNases and RNases of most species.
- Count on consistent quality and performance. Stringent quality testing ensures optimal stability and high-level lot-to-lot performance.
- Prepare samples over a wide range of conditions. The robust enzyme is stable over a wide pH range and is ideal for diverse applications.
- Benefit from a contamination-free enzyme. The enzyme is tested for the absence of RNases and DNases, and is virtually free of DNA. It is especially suited for the isolation of PCR templates.
This preparation is free of RNases, DNases, and DNA, according to the current quality control procedures.
Absence of Nucleases: Each lot is tested on various substrates to ensure the absence of endonuclease, exonuclease, ribonuclease, and nicking activity.
DNA Content: ≤10pg/mg enzyme (determined by Threshold)
Bioburden: ≤5cfu/g (determined by the most stringent test from the European Pharmacopoeia, which identifies the total number of viable aerobic bacteria, yeast, and fungi)
Volume Activity: Approximately 50 U/ml solution (chromozyme assay); approximately 600 U/ml solution (hemoglobin assay). One unit is the enzyme activity which cleaves at +25 °C in 1 min 18 mmol Chromozym TRY (equivalent to 600 U/ml with the hemoglobin assay). Refer to the Certificate of Analysis for specific values for the present lot.
Activator: To stimulate proteinase K activity, denaturing agents (SDS and urea) can be added. For example, SDS at a final concentration of 2% can increase the activity of proteinase K significantly. Optimization using denaturing agents can increase proteinase activity by as much as sevenfold.
Working solution: Suggested Buffers:
The most appropriate buffer for Proteinase K will vary from application to application. Always follow the pH and temperature guidelines in parameter filed. As a general rule, proteinase K is stable and very active in buffers that contain denaturing reagents such as urea, sodium dodecyl sulfate (SDS), and guanidinium salts.
Inhibitors: The enzyme is inactivated by Pefabloc® SC. However, it is not inactivated by metal ions, chelating agents (e.g., EDTA), sulfhydryl reagents, or trypsin and chymotrypsin inhibitors.
For the rapid inactivation of RNases and DNases.
The enzyme can reduce protein to free amino acids if it is present in large excess for long incubation periods. Proteinase kFor life science research only. Not for use in diagnostic procedures.