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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.
Recombinant Proteinase K, engineered to be completely heat inactivated at 55°C for just 10 minutes, while maintaining similar activity and specificity as the wild-type version.
No detectable endonuclease, exonuclease, DNase or RNase contaminating activities. Suitable for sequencing and amplification sample preparation as well as broad molecular biology applications.
Enables multi-step workflows in the same reaction vessel without the need for bead-based purification.
Robust activity across a wide range of reaction buffer types, salt concentrations, detergents, as well as DTT and EDTA, with optimal activity between 20 - 40°C and pH 7.0 - 9.5.
Thermolabile Proteinase K is a recombinantly engineered, subtilisin-related serine protease that will hydrolyze a variety of peptide bonds. Unlike other Proteinase K versions that can only be partially inactivated by heat, Thermolabile Proteinase K can be entirely heat-inactivated, allowing for simpler, more efficient workflows, without the need for additional purification steps to remove the enzyme. Thermolabile Proteinase K maintains similar activity and specificity to the wild type version, cleaving peptide bonds at the carboxyl side of most amino acid residues, with some preference at aliphatic or aromatic ones.
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: No remaining activity of Thermolabile Proteinase K after heat inactivation
NEB's Thermolabile Proteinase K can be completely (> 99.9%) heat inactivated at only 55°C for 10 minutes compared to wild-type Proteinase K with only partial inactivation, even at higher temperatures. Enzymes were diluted and heated in 20 mM Tris/HCl pH 7.5 and 1 mM of CaCl2, at concentrations that are typical to their respective reaction conditions.
Figure 2: Heat Inactivation of Thermolabile Proteinase K vs. other Proteinase K options
NEB’s Thermolabile Proteinase K can be completely heat inactivated at 55°C in minutes, while another supplier’s proteinase featuring heat-inactivation capabilities retains substantial residual activity after heated at 55°C or 60°C for 25 minutes.
Figure 3: 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.
Using Thermolabile Proteinase K instead of magnetic beads for cleanup in DNA or RNA library prep keeps the reaction in a single tube, streamlining workflows, improving yield, and reducing costs. This eliminates multiple wash steps, minimizes sample loss, and prevents enzyme carryover between steps. It's especially beneficial for low-input samples, automation, and high-throughput applications.
Product Source
Cloned from Engyodontium album (formerly Tritirachium album), mutagenized to increase thermolability of the enzyme and expressed in Pichia pastoris.
This product is related to the following categories:
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
P8111S
-20
Thermolabile Proteinase K
P8111SVIAL
-20
1 x 0.25 ml
120 units/ml
P8111L
-20
Thermolabile Proteinase K
P8111LVIAL
-20
1 x 1.25 ml
120 units/ml
Properties & Usage
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 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.
Advantages and Features
Features
Break down and inactivate proteins and enzymes with this unique Proteinase K that can be completely and rapidly heat inactivated. Effectively degrades all proteins, requires minimal enzyme for fast reactions, and functions across diverse conditions and workflows
Enables successive enzymatic reactions in a single reaction vessel without bead purification steps
Suitable for DNA, RNA extraction, nucleic acid sample preparation for sequencing and amplification applications
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.
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
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