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.
Reagents Supplied
The following reagents are supplied with this product:
|
NEB #
|
Component Name
|
Component #
|
Stored at (°C)
|
Amount
|
Concentration
|
Advantages and Features
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
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 min
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.
Related Products
Product Notes
-
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.
-
Thermolabile Proteinase K has the following activity in NEB specific restriction endonuclease buffers: 24% activity in NEBuffer 3.1, 37% activity in CutSmart Buffer, and 60% activity in NEBuffer1.1 and NEBuffer 2.1. In addition, the enzyme retains 80% activity in T4 DNA ligase buffer.
Selection Charts
Selection Charts
Quality Control Assays
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.
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]
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 following link is to a PDF Safety Data Sheet (SDS) that applies to this product to help you use it safely.
Legal and Disclaimers
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please email us at 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.