Product Class: Other

Thermolabile Proteinase K
cloned at NEB recombinant 55 Heat

This product is available in a glycerol-free format. Contact us for more information.

Product Introduction

This recombinant, heat-inactivated Proteinase K is engineered for complete inactivation at 55°C in 10 minutes, with broad protease activity for digestion of proteins in nucleic acid preparations and other applications.

  • A unique, recombinant Proteinase K that retains similar activity and specificity as wild type but can be completely heat inactivated at lower temperatures compared to others.
  • Engineered for complete heat inactivation with incubation at 55°C for only 10 minutes.
  • No detectable endonuclease, exonuclease, DNase or RNase contaminating activities.
  • 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.
  • Broad spectrum subtilisin-related serine protease that hydrolyzes a variety of peptide bonds and is frequently used to cleanup enzymatic reactions or cell lysates.
Catalog # Size Concentration
P8111S 30 units 120 units/ml

Product Information

Description

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 Pichia pastoris.
This product is related to the following categories:
Proteases Products,
Total RNA Extraction & Purification Products,
RNA Extraction and Purification,
Nucleic Acid Purification,
This product can be used in the following applications:
PCR & Reaction Cleanup,
Protein Analysis Tools,
Protein Digestion,
Proteomics,
DNA Amplification, PCR & qPCR

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

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.

Features

  • Heat inactivated following incubation at 55°C for 10 minutes.
  • Isolation of plasmid and genomic DNA and RNA
  • Inactivation of RNases, DNases and various other enzymes in reaction workflows
  • PCR purification

Product Notes

  1. 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.
  2. Thermolabile Proteinase K is stable for at least 2 years at –20°C. No loss of activity is observed after 10 freeze-thaw cycles.
  3. Thermolabile Proteinase K is highly active over a broad pH range (7.0 - 9.5) and a wide temperature range (20 – 40°C).
  4. Thermolabile Proteinase K is active in chelating agents such as EDTA up to 10 mM.
  5. 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%.
  6. Thermolabile Proteinase K activity is inhibited by urea concentrations greater than 2 M.

Protocols, Manuals & Usage

Protocols

  1. Thermolabile Proteinase K Typical Reaction Protocol

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. How is Thermolabile Proteinase K different from Proteinase K, Molecular Biology Grade?
  2. Can I use Thermolabile Proteinase K for molecular biology applications?
  3. Should I switch from Proteinase K, Molecular Biology Grade to Thermolabile Proteinase K?
  4. If I switch from Proteinase K, Molecular Biology Grade to Thermolabile Proteinase K, should I use the same amount of enzyme?
  5. How do I heat inactivate Thermolabile Proteinase K?
  6. What buffer should I use to dilute Thermolabile Proteinase K?
  7. What is the optimal reaction buffer for Thermolabile Proteinase K?
  8. What is the optimal incubation temperature and time?
  9. Is Thermolabile Proteinase K compatible with EDTA, Triton X-100, SDS, DTT and/or Urea?
  10. Is Thermolabile Proteinase K active in common NEB buffers?
  11. Is Thermolabile Proteinase K active in the presence of metal ions?
  12. 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)?
  13. What is the activity if Proteinase K, Molecular Biology Grade if subjected to the unit definition assay used for Thermolabile Proteinase K?

Quality, Safety & Legal

Quality Assurance Statement

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

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]

Certificate Of Analysis

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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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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