Thermolabile Proteinase K is an engineered, subtilisin-related serine protease that will hydrolyze a variety of peptide bonds and is frequently used to cleanup enzymatic reactions or cell lysates.
Heat inactivated following incubation at 55°C for 10 minutes.
Optimal activity and stability for up to 24 months.
Active in a wide range of reaction buffers with optimal activity between 20 - 40°C and pH 7.0 - 9.5.
No detectable endonuclease, exonuclease, DNase or RNase contaminating activities.
Thermolabile Proteinase K is an engineered, subtilisin-related serine protease that will hydrolyze a variety of peptide bonds. It preferentially cleaves the peptide bond at the carboxyl side of aliphatic or aromatic amino acid residues. However; the specificity of Thermolabile Proteinase K can be broad.
Thermolabile Proteinase K (TLPK) can be completely inactivated by incubation at 55°C for 10 minutes, which allows for subsequent enzymatic steps in the same reaction vessel. Figure 1 shows that the activity of restriction endonucleases, including heat-stable endonucleases, can be completely abolished using TLPK.
Figure 1: Thermolabile Proteinase K completely degrades restriction enzyme activity
Lane L: 1 kb DNA Ladder (NEB #N3232); Lane 1: λ DNA incubated for 1 hour at 37°C; Lane 2: λ DNA incubated with PvuII-HF or PstI-HF for 1 hour at 37°C; Lane 3: λ DNA incubated with PvuII-HF or PstI-HF which had been treated with 1 μL Thermolabile Proteinase K (TLPK) for 10 minutes at 37 °C; Lane 4: λ DNA incubated first with TLPK treated PvuII-HF or PstI-HF followed by incubation at 55 °C for 10 minutes to inactivate the TLPK, followed by treatment with PvuII-HF or PstI-HF for 1 hour at 37°C. Results indicate that Thermolabile Proteinase K completely degrades restriction enzyme activity, allowing for subsequent enzymatic steps in the same reaction vessel.
Product Source
Cloned from Engyodontium album (formerly Tritirachium album), mutagenized to increase thermolability of the enzyme and expressed in K. lactis.
This product is related to the following categories:
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 CaCl2, 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.
Active in a wide range of buffers. It is highly active between pH 7.0 and 9.5 and temperatures 20-40°C. It is active in chelating agents such as EDTA up to 10 mM.
Thermolabile Proteinase K is stable for at least 2 years at –20°C. No loss of activity is observed after 10 freeze-thaw cycles.
Thermolabile Proteinase K is highly active over a broad pH range (7.0 - 9.5) and a wide temperature range (20 – 40°C).
Thermolabile Proteinase K is active in chelating agents such as EDTA up to 10 mM.
Enzyme activity is stimulated in up to 1% SDS and no inhibition is observed in SDS concentrations up to 2.5% or Triton X-100 concentrations up to 1.5%.
Thermolabile Proteinase K activity is inhibited by urea concentrations greater than 2 M.
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