prou
Products
Proteinase K NGS (powder) HC4507A Featured Image
  • Proteinase K NGS (powder) HC4507A
  • Proteinase K NGS (powder) HC4507A

Proteinase K NGS (powder)


Cat No: HC4507A

Package: 1g/10g/100g/500g

 Free of DNase, RNase, Nickase

Activity: ≥40 U/mg

Nucleic Acid residue: ≤ 5 pg/mg

Bioburden: ≤ 50 CFU/g

Shelf life 3 years

Transportation in room temperature

One-batch capacity 30kg

 

Product Description

Product detail

Data

Cat No: HC4507A

NGS Protease K is a stable serine protease with high enzyme activity and wide substrate specificity.The enzyme preferentially decomposes ester bonds and peptide bonds adjacent to the C-terminal of hydrophobic amino acids, sulfur-containing amino acids and aromatic amino acids. So, it is often used to degrade proteins into short peptides. NGS Protease K is a typical serine protease with the Asp39-His69-Ser224 catalytic triad which is unique to serine proteases, and the catalytic center is surrounded by tow Ca2+ binding sites for stabilization, maintaining high enzyme activity under a wider range of conditions.


  • Previous:
  • Next:

  • Specification

    Appearance

    White to off-white amorphous powder, lyophilized

    Specific activity

    ≥40U/mg solid

    DNase

    None detected

    RNase

    None detected

    Bioburden

    ≤50CFU/g solid

    Nucleic acid residue

    <5pg/mg solid

     

    Properties

    Source

    Tritirachium album

    EC number

    3.4.21.64 (Recombinant from Tritirachium album)

    Molecular weight

    29kDa (SDS-PAGE)

    Isoelectric point

    7.81                                                                                              Fig.1

    Optimum pH

    7.0-12.0 (All perform high activity)                                            Fig.2

    Optimum temperature

    65℃                                                                                             Fig.3

    pH Stability

    pH 4.5-12.5 (25℃,16h)                                                               Fig.4

    Thermal stability

    Below 50℃ (pH 8.0, 30min)                                                       Fig.5

    Storage stability

    Stored at 25℃ for 12months                                                       Fig.6

    Activators

    SDS, urea

    Inhibitors

    Diisopropyl fluorophosphate; benzylsulfonyl fluoride

     

    Storage Conditions

    Store the lyophilized powder at -25~-15 ℃ for a long time away from light; After dissolution, aliquot into appropriate volume for short-term storage at 2-8℃ away from light or long-term storage at -25~-15 ℃ away from light.

     

    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.

     

    Unit definition

    One unit of NGS Protease K is defined as the amount of enzyme required to hydrolyze casein into 1 μmol L-tyrosine under standard determination conditions.

     

     Reagents preparation

    Reagent

    Manufacturer

    Catalog

    Casein technical from bovine milk

    Sigma Aldrich

    C7078

    NaOH

    Sinopharm Chemical Reagent Co., Ltd.

    10019762

    NaH2PO4 ·2H2O

    Sinopharm Chemical Reagent Co., Ltd.

    20040718

    Na2HPO4

    Sinopharm Chemical Reagent Co., Ltd.

    20040618

    Trichloroacetic acid

    Sinopharm Chemical Reagent Co., Ltd.

    80132618

    Sodium acetate

    Sinopharm Chemical Reagent Co., Ltd.

    10018818

    Acetic acid

    Sinopharm Chemical Reagent Co., Ltd.

    10000218

    HCl

    Sinopharm Chemical Reagent Co., Ltd.

    10011018

    Sodium carbonate

    Sinopharm Chemical Reagent Co., Ltd.

    10019260

    Foline-phenol

    Sangon Biotech (Shanghai) Co., Ltd.

    A500467-0100

    L-tyrosine

    Sigma

    93829

    Reagent I:

    Substrate: 1% Casein from bovine milk solution: dissolve 1g bovine milk casein in 50ml of 0.1M sodium phosphate solution, pH 8.0, heat in water bath at 65-70 °C for 15mins, stir and dissolve, cool with water, adjusted by sodium hydroxide to pH 8.0, and dilute in to 100ml.

    Reagent II:

    TCA solution: 0.1M trichloroacetic acid, 0.2M sodium acetate and 0.3M acetic acid (weigh 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid successively, add 50mL deionized water, adjust with HCl to pH 4.03, and dilute into 100ml).

    Reagent III:

    0.4m sodium carbonate solution (weigh 4.24g anhydrous sodium carbonate and dissolve in 100mL water)

    Reagent IV:

    Folin phenol reagent: dilute 5 times with deionized water.

    Reagent V:

    Enzyme diluent: 0.1 M sodium phosphate solution, pH 8.0.

    Reagent VI:

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

     

    Procedure

    1. Turn on the UV-Vis spectrophotometer and select photometric measurement.

    2. Set the wavelength as 660nm.

    3. Turn on the water bath, set the temperature to 37℃, ensure the temperature unchanged for 3-5mins.

    4. Preheat 0.5mL substrate in a 2mL centrifuge tube at 37℃ water bath for 10mins.

    5. Extract 0.5mL diluted enzyme solution into the preheated centrifuge tube for 10 minutes. Set enzyme diluent as blank group.

    6. Add 1.0 mL TCA reagent immediately after the reaction. Mix well and incubate in water bath for 30 minutes.

    7. Centrifugate reaction solution.

    8. Add the following components in the order specified.

    Reagent

    Volume

    Supernatant

    0.5 mL

    0.4M Sodium carbonate

    2.5 mL

    Folin phenol reagent

    0.5 mL

    9. Mix well before incubating in water bath a 37℃ for 30 mins.

    10. OD660 was determined as OD1; blank control group: Enzyme diluent is used to replace enzyme solution to determine OD660 as OD2, ΔOD=OD1-OD2.

    11. L-tyrosine standard curve: 0.5mL different concentration L-tyrosine solution, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent 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)

    Figures

     

    Fig.1 DNA residue

    Sample

    Ave C4

    Nucleic Acid

    Recovery(pg/mg)

    Recovery(%)

    Total Nucleic

    Acid ( pg/mg)

    PRK

    24.66

    2.23

    83%

    2.687

    PRK+STD2

    18.723

    126.728

    STD1

    12.955

     

     

     

     

     

     

     

     

     

     

     

     

    STD2

    16

    STD3

    19.125

    STD4

    23.135

    STD5

    26.625

    RNA-Free H2O

    Undetermined

     

    Fig.2 Optimum pH

     

    Fig.3 Optimum temperature

     

    Fig.4 pH Stability

     

    Fig.5 Thermal stability

     

    Fig.6 Storage stability at 25℃

     

     

     

     

    Write your message he