Thermolabile Proteinase K |NEB酶试剂 New England Biolabs

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产品信息

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

Thermolabile Proteinase K |

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.

产品来源

Cloned from Engyodontium album (formerly Tritirachium album), mutagenized to increase thermolability of the enzyme and expressed in Pichia pastoris.

产品类别:
Proteases Products,
Total RNA Extraction & Purification Products,
RNA Extraction and Purification,

Nucleic Acid Purification Products

应用:
PCR & Reaction Cleanup,
Protein Analysis Tools,
Protein Digestion,

Proteomics,

DNA Amplification, PCR & qPCR

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • P8111S     -20    
        Thermolabile Proteinase K P8111SVIAL -20 1 x 0.25 ml 120 units/ml

  • 特性和用法

    单位定义

    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.

    贮存溶液

    20 mM Tris-HCl
    1 mM CaCl2
    50% Glycerol
    pH 7.4 @ 25°C

    热失活

    55°C for 10 minutes

    分子量

    实际: 29 kDa

    单位活性检测条件

    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.

  • 优势和特性

    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

  • 相关产品

    相关产品

    • p8107-proteinase-k-molecular-biology-grade

  • 注意事项

    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.

操作说明、说明书 & 用法

  • 操作说明

    1. Thermolabile Proteinase K Typical Reaction Protocol

工具 & 资源

  • 选择指南

    • Protease Selection Chart

FAQs & 问题解决指南

  • 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?