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  • Proteinase K (liquid)

Proteinase K( Liquid)


Cat No:HC4502A

Package:5ml/100ml/1L/10L

Proteinase K is a stable serine protease with broad substrate specificity. It degrades many proteins in the native state even in the presence of detergents.

Product Description

Product detail

Data

Proteinase K is a stable serine protease with broad substrate specificity. It degrades many proteins in the native state even in the presence of detergents. Evidence from crystal and molecular structure studies indicates the enzyme belongs to the subtilisin family with an active site catalytic triad (Asp39-His69- Ser224). The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity.


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  • Specification

    Appearance

    Colorless to light brown liquid

    Activity

    ≥800 U/ml

    Protein concentration

    ≥20 mg/ml

    DNase

    None detected

    RNase

    None detected

     

    Storage Conditions

    Store at temperature of 2-8℃.

     

    Properties

    EC number

    3.4.21.64 (Recombinant from Tritirachium album)

    Molecular weight

    29 kDa (SDS-PAGE)

    Isoelectric point

    7.81

    Optimum pH

    7.0-12.0                                                                                        Fig.1

    Optimum temperature

    65 ℃                                                                                            Fig.2

    pH stability

    pH 4.5-12.5(25℃, 16 h)                                                              Fig.3

    Thermal stability

    Below 50℃ (pH 8.0, 30 mins)                                                    Fig.4

    Activators

    SDS, urea

    Inhibitors

    Diisopropyl fluorophosphate; phenylmethylsulfonyl fluoride

     

    Applications

    1. Genetic diagnostic kit

    2. RNA and DNA extraction kits

    3. Extraction of non-protein components from tissues, degradation of protein impurities, such as DNA vaccines and preparation of heparin

    4. Preparation of chromosome DNA by pulsed electrophoresis

    5. Western blot

    6. Enzymatic glycosylated albumin reagents in vitro diagnosis

     

    Precautions

    Wear protective gloves and goggles when using or weighing, and keep well ventilated after use. This product may cause skin allergic reaction and serious eye irritation. If inhaled, it may cause allergy or asthma symptoms or dyspnea. May cause respiratory irritation.

     

    Assay

    Unit definition

    One unit (U) is defined as the amount of enzyme required to hydrolyze casein to produce 1 μmol tyrosine per minute under the following conditions.

     

    Reagents preparation

    Reagent I:1 g milk casein was dissolved in 50 ml of 0. 1 M sodium phosphate solution (pH 8.0), incubated in 65-70 ℃ water for 15mins, stirred and dissolved, cooled by water, adjusted by sodium

    hydroxide to pH8.0, and fixed volume 100ml.

    Reagent II:TCA solution:0. 1 Mtrichloroacetic acid, 0.2 M sodium acetate, 0.3 M acetic acid.

    Reagent III:0.4 MNa2CO3 solution.

    Reagent IV:Forint reagent diluted with pure water for 5 times.

    Reagent V:enzyme diluent:0. 1 M sodium phosphate solution (pH 8.0).

    Reagent VI:tyrosine solution:0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine dissolved with 0.2M HCl.

     

    Procedure

    1.0.5 ml of reagent I is pre-warmed to 37℃, add 0.5 ml of enzyme solution, mix well, and incubate at 37℃ for 10mins.

    2. Add 1 ml of reagent II to stop the reaction, mix well, and continue incubation for 30mins.

    3. Centrifugate reaction solution.

    4. Take 0.5 ml supernatant, add 2.5 ml reagent III, 0.5 ml reagent IV, mix well and incubate at 37℃ for 30mins.

    5. OD660 was determined as OD1 ; blank control group: 0.5 ml reagent V is used to replace enzyme solution to determine OD660 as OD2, ΔOD=OD1-OD2.

    6. L-tyrosine standard curve: 0.5mL different concentration L-tyrosine solution, 2.5mL Reagent III, 0.5mL Reagent IV in 5mL centrifuge tube, incubate in 37℃ for 30mins, detect for OD660 for different concentration of L-tyrosine, then obtained the standard curve Y=kX+b, where Y is the L-tyrosine concentration, X is OD600.

     

     Calculation

     

    2: Total volume of reaction solution (mL)

    0.5: Volume of enzyme solution (mL)

    0.5: Reaction liquid volume used in chromogenic determination (mL)

    10: Reaction time (min)

    Df: Dilution multiple

    C: Enzyme concentration (mg/mL)

     

    References

    1. Wieger U & Hilz H. FEBS Lett. (1972); 23:77.

    2. Wieger U & Hilz H. Biochem. Biophys. Res. Commun. (1971); 44:513.

    3. Hilz, H. et al., Eur. J. Biochem. (1975);56:103–108.

    4. Sambrook J et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(1989).

     

    Figures

    Fig. 1 Optimum pH

    100mM buffer solution: pH6.0-8.0, Na-phosphate; pH8.0- 9.0, Tris-HCl; pH9.0-12.5, Glycine-NaOH.Enzyme concentration:1mg/mL

     

    Fig. 2 Optimum temperatur

    Reaction in 20 mM K-phosphate buffer pH 8.0.Enzyme concentration:1mg/mL

     

    Fig. 3 pH Stability

    25℃,16 h-treatment with 50 mM buffer solution: pH 4.5-5.5, Acetate; pH 6.0-8.0, Na-phosphate; pH 8.0-9.0, Tris-HCl. pH 9.0-12.5, Glycine-NaOH.Enzyme concentration:1mg/mL

     

    Fig. 4 Thermal stability

    30 min-treatment with 50mM Tris-HCl buffer, pH 8.0.Enzyme concentration:1mg/mL

     

    Fig. 5 Storage stability at 25℃

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