Proteinase K is a subtilisin-related serine protease that hydrolyzes a variety of peptide bonds and is frequently used to cleanup enzymatic reactions or cell lysates.
Highly characterized for more consistent performance
Optimal activity and stability for up to 24 months
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.
No detectable endonuclease, exonuclease, DNase or RNase contaminating activities
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).
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. Figure 2: 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)
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
P8107S
-20
Proteinase K, Molecular Biology Grade
P8107SVIAL
-20
2 x 1 ml
800 units/ml
Properties & 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 Buffer
20 mM Tris-HCl
1 mM CaCl2
50% Glycerol
pH 7.4 @ 25°C
Heat Inactivation
95°C for 10 minutes
Molecular Weight
Theoretical: 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.
Advantages and Features
Application Features
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
Product Notes
Active in a wide range of buffers, including all NEB-specific restriction endonuclease buffers. It is highly active between pH 7.5 and 12 and temperatures 20-60°C. Proteinase K is also active in chelating agents such as EDTA and activity is stimulated in up to 2% SDS or 4M urea.
Proteinase K is stable for at least 2 years at –20°C. No loss of activity is observed after 10 freeze-thaw cycles.
Reaction Conditions Proteinase K is active in a wide range of buffers including all NEB specific restriction endonuclease buffers. It is highly active between pH 7.5 and 12.0 and temperatures between 20 and 60°C (1,2). Proteinase K is also active in chelating agents such as EDTA (4) and activity is stimulated in up to 2% SDS or 4 M urea (3).
References
Bajorath, J. et al. (1988). Biochimica et Biophysica Acta. 954, 176-182.
Ebeling, W. et al. (1974). Eur. J. Biochem. 47, 91-97.
Hilz, H. et al. (1975). Eur. J. Biochem. 56, 103-108.
Bajorath, J. et al. (1988). Eur. J. Biochem. 176, 441-447.
Crowe, J.S. et al, (1991). Nucleic Acids Research. 91, 184.
Anson, M.L. (1939). J. Gen. Physiol. 22, 79-89.
Pace, C.N. et al. (1995). Protein Sci. 4, 2411-2423.
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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