Seebio’s Proteinase K
Time：2023-12-05 Hits：223
Proteinase K is a highly active protease belonging to the serine protease class, used for the general degradation of proteins in biological samples. It is purified from Tritirachium album limber, a strain of the fungus Tritirachium album. Proteinase K is a serine protease with broad cleavage activity, cutting peptide bonds at the carboxyl end of aliphatic and aromatic amino acids. This enzyme has been purified to eliminate RNA and DNA nuclease activities. Due to its stability in urea and SDS, Proteinase K also possesses the ability to degrade native proteins.
Applications of Proteinase K:
Gene diagnostic kits
Genomic DNA extraction kits
Nuclease removal in RNA extraction kits for DNA and RNA preparation
Degradation of non-protein components in tissue extraction containing proteinaceous impurities, such as in the preparation of DNA vaccines and heparin
Preparation of chromosomal DNA for pulse electrophoresis
Protein blotting
The general working concentration of Proteinase K is 50-100 μg/ml.
Proteinase K Indicatornase K indicator
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Product name
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Proteinase K
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Source
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Tritirachium album limber
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Exterior
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White freeze-dried powder
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Specific activity
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>30U/mg protein
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Molecular weight
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29,730
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CAS No.
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39450-01-6
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EC No
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3.4.21.14
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Activity unit
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37℃ Under the condition of pH 7.5, the amount of proteinase K that can hydrolyze the substrate casein to produce 1 μmol of tyrosine per minute is defined as one unit (U）。
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DNase residue
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Using λDNA as the substrate and digesting it at 37°C for more than 6 hours, no deoxyribonuclease activity was detected.。
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RNase residue
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Digestion at 25℃ for more than 16 hours, no ribonuclease activity was detected.。
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Deactivation method
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Adding PMSF or DFP inhibitor to the reaction system can inactivate it, and incubating it at 65°C for 10-15 minutes can partially inactivate it.
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Package
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100mg, 1g, 10g, 1kg large packaging inquiry
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Proteinase K storage
The enzyme can be stored in a dry state at 0-4°C. After reconstitution, it should be aliquoted into suitable volumes and stored at -20°C. Transportation can be conducted at room temperature. Under proper storage conditions, the shelf life of the enzyme in dry form is up to one year. In liquid form, it is effective for six months at -20°C. After opening the package, if left at 2-8°C for more than a week, it is recommended to filter for sterility or add stabilizers such as 0.5% sodium azide to prevent microbial contamination.
Storage and Dilution Buffer: 50 mM Tris-HCl buffer, 2 mM CaCl2 (pH 8.0).
Reaction Conditions: Incubate at 37-56°C for one hour in the reaction buffer.
Proteinase K Experimental Data
Enzyme Activity Assay and Stability Test
(1) Enzyme Activity Assay
Reagent Preparation
Reagent I - Substrate: 1% casein solution from bovine milk.
Dissolve 1 g of casein in 50 ml of 0.1 M sodium phosphate buffer, pH 8.0, at 65-70°C with stirring for 15 minutes. Cool the solution with tap water, adjust pH to 8.0 with sodium hydroxide, and make up to 100 ml.
Reagent II - TCA Solution: 0.1 M trichloroacetic acid, 0.2 M sodium acetate, 0.3 M acetic acid. Adjust pH to 4.03 with HCl and make up to 100 ml.
Reagent III - 0.4 M sodium carbonate solution.
Reagent IV - Folin phenol reagent: Dilute 5 times with water.
Reagent V - Enzyme Dilution Solution: 0.1 M sodium phosphate buffer, pH 8.0.
Reagent VI - Tyrosine Solution: Dissolve 1 μg/ml tyrosine in 0.2 M HCl.
Experimental Steps
1. Incubate 0.5 ml of Reagent I at 37°C for 10 minutes, add 0.5 ml of enzyme solution, mix well, and react at 37°C for 10 minutes.
2. Add 1 ml of Reagent II to terminate the reaction, mix well, and continue incubation for 30 minutes.
3. Centrifuge the reaction mixture.
4. Take 0.5 ml of the supernatant, add 2.5 ml of Reagent III and 0.5 ml of Reagent IV, mix well, and incubate at 37°C for 30 minutes.
5. Measure OD1 at 660 nm; Blank control group: Substitute 0.5 ml of Reagent V for the enzyme solution and measure OD2.
6. Take 0.5 ml of Reagent VI, 2.5 ml of Reagent III, and 0.5 ml of Reagent IV, mix well, and incubate at 37°C for 30 minutes. Measure OD3 at 660 nm;
Blank control group: Substitute 0.5 ml of 0.2 M HCl for Reagent VI, and the measured value is OD4.
Enzyme Activity Calculation
Formula Description：
2: Total volume of the reaction mixture (in mL). This represents the overall volume of the reaction environment where the enzymatic activity is taking place.
0.5: Volume of the enzyme solution (in mL). This is the amount of the enzyme solution added to the reaction mixture.
10: Reaction time (in minutes). This indicates the duration for which the enzymatic reaction is allowed to occur.
df: Dilution factor. This factor accounts for any dilution of the enzyme during the experimental procedure.
181.2: Molecular weight of tyrosine (in g/mol). This is the molecular mass of the amino acid tyrosine.
C: Enzyme concentration (in mg/mL). This denotes the concentration of the enzyme in the experimental setup.
Compare results
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Proteinase K specific activity
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manufacturer
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Foreign M brand
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Seebio
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Lyophilized product activity u/mg
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33.5
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49.5
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(2) Purity and Protein Digestion Test (SDS-PAGE)
Results: Protein purity exceeds 99% (confirmed by laser scanning SDS-PAGE gel).
(3) Stability Test:
Conclusion: The lyophilized powder maintains consistent activity when stored correctly for up to one year; the liquid enzyme shows a decrease in activity of less than 5% within six months.
2. Comparative Test
Comparison between Roche Proteinase K and Seebio Proteinase K.
Comparison Reaction Conditions: Roche Proteinase K: 65°C for 10 minutes; Seebio Proteinase K: 65°C for 10 minutes; Seebio Proteinase K: 55°C for 60 minutes
Results from quality control (QC) meet the requirements. Each tube contains 500 μl of whole blood, with a total of three control groups.
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Experimental conditions
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Yield
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ROCHE65℃10m
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33ug
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19.2ug
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5ug
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SEEBIO65℃10m
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30ug
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20ug
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4.8ug
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SEEBIO55℃60m
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30.4ug
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18.5ug
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5.4ug
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Whole blood DNA extraction kit electrophoresis chart
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DNA actin electrophoresis pattern extracted from whole blood
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date：2010.06.09
Gel concentration: 0.7% agarose RNA gel Loading volume: 1ug/well Electrophoresis conditions: U=105V T=15min 1. 090826-15（Roche, 65℃ 10m） 2. 090826-15（seebio, 65℃ 10m） 3. 090826-15（seebio, 55℃ 60m） 4. 090826-17（Roche, 65℃ 10m） 5. 090526-17（seebio, 65℃ 10m） 6. 090826-17（seebio, 55℃ 60m） 7. 090826-20（Roche, 65℃ 10m） 8. 090826-20（seebio, 65℃ 10m） 9. 090826-20（seebio, 55℃ 60m） |
date：2010.06.10
Gel concentration: 0.7% agarose DNA gel Loading volume: 10ug/well Electrophoresis conditions: U=95V T=15min 1. 090826-15（Roche, 65℃ 10m） 2. 090826-15（seebio, 65℃ 10m） 3. 090826-15（seebio, 55℃ 60m） 4. 090826-17（Roche, 65℃ 10m） 5. 090526-17（seebio, 65℃ 10m） 6. 090826-17（seebio, 55℃ 60m） 7. 090826-20（Roche, 65℃ 10m） 8. 090826-20（seebio, 65℃ 10m） 9. 090826-20（seebio, 55℃ 60m） －：Negative control ＋：Positive Control |
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Conclusion: The experimental results comparing SEEBIO Proteinase K with Roche Proteinase K show similar yields and gel electrophoresis patterns after a 10-minute incubation at 65°C. The effects are essentially the same, indicating that SEEBIO Proteinase K can be used as a substitute for Roche Proteinase K.
Proteinase K Product List
Proteinase KProduct List
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Item number
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Product name
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Packaging specifications
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EBY0027M
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Proteinase K (lyophilized, water-soluble）
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100mg, 1g
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EBY0027J
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Proteinase K (lyophilized, glycerol-soluble）
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100mg、1g
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Refereces:
1. Kraus, E; et.al. Proteinase K from the Mold Tritirachium album limber, Specificity and Mode of Action. Z. Physiol. Chem., 357:939; 1976.
2. Jany,KD, et al. Amino Acid Sequence of Proteinase K from the Mold, Tritirachium album limber. Proteinase K; a Subtilisin-related Enzyme with Disulfide Bonds. FEBS Letter, 199,139.1986.
3. Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Volume 3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, B.16.
4. Sweeney, PJ and Walker, JM (1993) Enzymes of molecular biology. In: Methods in Molecular Biology, Vol. 16, MM Burrell, ed., Humana Press, Inc., Totowa, NJ, 305.