New England Biolabs, P8111S, Thermolabile Proteinase K

Thermolabile Proteinase K is a recombinant Proteinase K engineered for rapid and complete heat inactivation with broad protease activity, for protein and enzyme degradations in nucleic acid preparations and other applications. Related Categories Proteases,, Total RNA Extraction & Purification,, PCR, qPCR & Amplification Technologies, Applications PCR & Reaction Cleanup,, Protein Analysis Tools,, Protein Digestion, Specification Unit Definition One unit is defined as the amount of enzyme required to release 1.0 µmol of 4-nitroaniline per minute from N-Succinyl -Ala-Ala-Pro-Phe-p-nitroanilide at 25°C, in a total reaction volume of 105 μl. Storage Buffer 20 mM Tris-HCl 1 mM CaCl2 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 55°C for 10 minutes Molecular Weight Apparent: 29 kDa Unit Assay Conditions A series of dilutions of Thermolabile Proteinase K are incubated with 0.25 mM N-Succinyl -Ala-Ala-Pro-Phe-p-nitroanilide in 0.1% SDS, 0.1% Triton X-100, 20 mM Tris-HCl, 5 mM CaCl 2 , 50 mM NaCl (pH 8.0 @ 25°C) in a 105 μl reaction. The reaction mix is incubated at 25°C. Liberation of p-nitroaniline is detected by real-time UV spectroscopy at 405 nm. FAQ Q: How is Thermolabile Proteinase K different from Proteinase K, Molecular Biology Grade? A: Thermolabile Proteinase K is an engineered and recombinantly expressed variant of Proteinase K, Molecular Biology Grade (wild type). The cleavage patterns of both enzymes are the same: they prefer aromatic and aliphatic amino acids. Thermolabile Proteinase K can be heat inactivated by incubating at 55°C for 10 minutes; and it retains optimal activity between 20–40°C. Q: Can I use Thermolabile Proteinase K for molecular biology applications? A: Thermolabile Proteinase K is free of exo- and endonuclease activity, RNase activity, and contains no detectable DNA (both bacterial and eukaryotic) contamination. Therefore, it is ideally suited for molecular biology and genomic research applications. Q: Should I switch from Proteinase K, Molecular Biology Grade to Thermolabile Proteinase K? A: If the experiment and/or application requires inactivation of Proteinase K to enable downstream reactions then yes, Thermolabile Proteinase K is the ideal option. Using Thermolabile Proteinase K allows one to avoid performing phenol/chloroform extraction or gel/column purification. Q: If I switch from Proteinase K, Molecular Biology Grade to Thermolabile Proteinase K, should I use the same amount of enzyme? A: For most applications, the same volume of Thermolabile Proteinase K can be used in place of Proteinase K, Molecular Biology Grade. This is a good starting point; however, the exact amount of enzyme required may be substrate dependent and may need to be tested empirically. Q: How do I heat inactivate Thermolabile Proteinase K? A: For the majority of workflows, Thermolabile Proteinase K can be heat inactivated by incubating at 55°C for 10 minutes. However, the thermolability of the enzyme is affected by salt concentration, pH, and the concentration of metal ions present in the reaction mixture. Therefore, the optimal inactivation temperature and incubation time may need to be empirically determined for specific workflows. Q: What buffer should I use to dilute Thermolabile Proteinase K? A: The amount of Thermolabile Proteinase K required may be substrate dependent. However, typical reaction conditions are 1 µl enzyme / 50 μl reaction volume. A recommended dilution buffer is 20 mM Tris-HCl, 1 mM CaCl2, pH 7.5 Q: What is the optimal reaction buffer for Thermolabile Proteinase K? A: Thermolabile Proteinase K is compatible with a wide range of buffers: Optimal pH: 7–10 Salt: 0–1 M NaCl Buffer: Tris-HCl, HEPES, Tricine, and Tris-Acetate. The table below shows relative activities in common buffers. 1X Buffer NEB1.1 + NEB2.1 + NEB3.1 + CutSmart +++ Q5 Buffer +++ Q5 HighGC + Thermopol + T4 DNA Ligase ++ Q: What is the optimal incubation temperature and time? A: The recommended incubation temperate range is 20–40˚C. For incubations longer than 1 hour, we recommend 25˚C. The incubation time is substrate dependent and varies based on the reason for proteolysis. Typically, to eliminate enzyme activity, a 5–10 minute incubation is adequate. However, it may take longer if cells need to be lysed, nucleosomes need to be degraded, histones need to be cleaved, or if other proteins are present. Q: Is Thermolabile Proteinase K compatible with EDTA, Triton X-100, SDS, DTT and/or Urea? A: Yes. Thermolabile Proteinase K is fully active in the presence of 10 mM EDTA, 1.5% Triton X-100, 2.5% SDS, 10 mM DTT, and 1M Urea. We recommend staying at or below these amounts of each component. Q: Is Thermolabile Proteinase K active in common NEB buffers? A: Yes, Thermolabile Proteinase K is active in NEBuffer 1.1, 2.1, 3.1, CutSmart®, and T4 DNA Ligase Reaction Buffer. Other buffers will need to be tested empirically. Q: Is Thermolabile Proteinase K active in the presence of metal ions? A: Thermolabile Proteinase K is active in the presence of 10mM CaCl2 and 10 mM MgCl2. Q: Why is the unit definition assay for Thermolabile Proteinase K (NEB #P8111) different from the unit definition assay for Proteinase K, Molecular Biology Grade (NEB # P8107)? A: Proteinase K, Molecular Biology Grade (#P8107) uses a traditional denatured hemoglobin substrate to measure activity (1). This traditional assay allows comparison of Proteinase K activity to the current and historical market. Over the years, casein has also been used as a substrate with less precision then hemoglobin. Thermolabile Proteinase K (#P8111) uses a more modern approach in which a nitroanilide substrate is used to measure activity by visible absorbance. This assay is preferred for its direct readout of proteolysis, sensitivity and 96-well plate compatibility. For the development of the direct assay see Pozgay et al. (2). References Anson, M. L. et al. (1938) J. Gen. Physiol., 22, 78-89. “The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin.” Pozsgay, M. et al. (1979) Eur. J. Biochem., 95, 115-119 “A method for designing peptide substrates for proteases. Tripeptidyl-p-nitroanilide substrates for Subtilisin Carlsberg” Q: What is the activity if Proteinase K, Molecular Biology Grade if subjected to the unit definition assay used for Thermolabile Proteinase K? A: Proteinase K, Molecular Biology Grade yields an activity concentration of 800 U/mL using the traditional denatured hemoglobin activity assay. The same sample yields an activity concentration of ~4,000 U/mL using the more modern nitroanilide substrate visible absorbance activity assay that is used for Thermolabile Proteinase K.