• My NEB
  • Print
  • PDF
  • Proteinase K, Molecular Biology Grade

    Catalog #SizeConcentrationPriceQtyAdd to Cart
    P8107S2 ml800 units/ml$67.00Add to Cart
      
    Categories:
    Proteases

    Description

    Highly characterized for more consistent performance, Proteinase K is a subtilisin-related serine protease that will hydrolyze a variety of peptide bonds. Proteinase K is active in a wide range of temperatures and buffers with optimal activity between 20 and 60°C and a pH between 7.5 and 12.0 (1, 2). Activity is stimulated when up to 2% SDS or up to 4 M urea are included in the reaction (3). Calcium is important for thermostability of Proteinase K but it is not required for catalysis, therefore Proteinase K is also active in buffers containing chelating agents such as EDTA (4).

    Lot-to-Lot Variability: Competitor Comparison
    Activity measurements from three NEB lots of Proteinase K were compared to three lots from another vendor. Reported activity for all NEB lots is 800 Units/ml ± 10% (indicated as a black line with error bars). Direct measurement of the competitor’s material using an assay reported on the vendor’s literature, demonstrated a dramatic over-assessment of all three lots.

    Activity Measurements: Competitor Comparison
    Activity measurements from a single NEB lot were compared to a single lot from seven other vendors. Proteinase K from NEB is sold at 800 Units/ml ± 10% (indicated as a black line with error bars), whereas most other vendors sell Proteinase K as > 800 or > 600 Units/ml. While Proteinase K from some vendors was close to the stated activity values, others varied significantly from the expected amount.

    Protein Concentration

    20 mg/ml, approximately, as determined by UV absorption at 280 nm.

    Product Source

    Engyodontium album (Tritirachium album)

    Advantages and Features

    Applications

    • Isolation of plasmid and genomic DNA 
    • Isolation of RNA
    • Inactivation of RNases, DNases and enzymes in reactions
    • Removal of enzymes from DNA to improve cloning efficiency (5)
    • PCR purification

    Properties and Usage

    Unit Definition

    One unit will digest urea-denatured hemoglobin at 37°C (pH 7.5) per minute to produce equal absorbance as 1.0 μmol of L-tyrosine using Folin & Ciocalteu's phenol reagent (6).

    Storage Temperature

    -20°C

    Storage Conditions

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

    Molecular Weight

    Calculated: 28.9 kDa

    Unit Assay Conditions

    0.5–2 μg of Proteinase K is incubated with 2% denatured hemoglobin solution for 10 minutes at 37°C (pH 7.5). After precipitation, neutralization and addition of Folin & Ciocalteu's phenol reagent, absorbance of soluble cleavage products are measured at 750 nm. Absorbance is compared to a standard curve of L-tyrosine absorbance prepared similarly.

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • qPCR DNA Contamination (eukaryotic genomic):
      The product is screened for the presence of eukaryotic genomic DNA using SYBR® Green qPCR with primers specific to the eukaryotic 18S rRNA locus. Results are quantified using a standard curve generated from purified Engyodontium album genomic DNA.
    • RNase Activity (Extended Digestion):
      The product is tested in a reaction containing a RNA substrate. After incubation for 16 hours greater than 90% of the substrate RNA remains intact as determined by gel electrophoresis.
    • Single Stranded DNase Activity (FAM Labeled Oligo):
      The product is tested in a reaction containing a fluorescent internal labeled single stranded oligonucleotide. The percent degradation is determined by capillary electrophoresis.

    Notes

    1. Proteinase K is stable for at least 2 years at –20°C. No loss of activity is observed after 10 freeze-thaw cycles.

    2. Reaction Conditions
      Proteinase K is active in awide range of buffers including all NEB specificrestriction endonuclease buffers. It is highly activebetween pH 7.5 and 12.0 and temperatures between20 and 60°C (1,2). Proteinase K is also active inchelating agents such as EDTA (4) and activity isstimulated in up to 2% SDS or 4 M urea (3).

    References

    1. Bajorath, J. et al. (1988). Biochimica et Biophysica Acta. 954, 176-182.
    2. Ebeling, W. et al. (1974). Eur. J. Biochem. 47, 91-97.
    3. Hilz, H. et al. (1975). Eur. J. Biochem. 56, 103-108.
    4. Bajorath, J. et al. (1988). Eur. J. Biochem. 176, 441-447.
    5. Crowe, J.S. et al, (1991). Nucleic Acids Research. 91, 184.
    6. Anson, M.L. (1939). J. Gen. Physiol. 22, 79-89.
    7. Pace, C.N. et al. (1995). Protein Sci. 4, 2411-2423.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. How is Proteinase K, Molecular Biology Grade (P8107) different from the previous Proteinase K product (P8102)?
    2. Why was the previous Proteinase K product (P8102) discontinued?
    3. Can I swap the new product into my existing protocol?
    4. What buffer should I use for Proteinase K?
    5. If I need to dilute Proteinase K, Molecular Biology Grade, how should I do this?
    1. General protocol to release nucleic acids prior to capillary or gel electrophoresis using Proteinase K (P8107)
    2. Protocol to cleanup DNA Glucosylation/restriction digest and Proteinase using Proteinase K (P8107)
    3. Protocol to purify PCR products in preparation for cloning using Proteinase K (P8107)