Proteinase K NGS (ufa)
Nambala ya mphaka: HC4507A
NGS Protease K ndi serine protease yokhazikika yokhala ndi ma enzyme ambiri komanso kukhazikika kwa gawo lapansi. The enzyme imakonda kuwola zomangira za ester ndi peptide zoyandikana ndi C-terminal ya hydrophobic amino acid, ma amino acid okhala ndi sulfure komanso ma amino acid onunkhira.Chifukwa chake, nthawi zambiri amagwiritsidwa ntchito kusokoneza mapuloteni kukhala ma peptides amfupi.NGS Protease K ndi mtundu wa serine protease wokhala ndi Asp39-Wake69-Ser224catalytic triad yomwe ili yapadera kwa serine proteases, ndipo malo othandizira azunguliridwa ndi tow Ca.2+malo omangira kuti akhazikike, kusunga ntchito yayikulu ya enzyme pansi pamikhalidwe yochulukirapo.
Kufotokozera
| Maonekedwe | White mpaka woyera amorphous ufa, lyophilized |
| Zochitika zenizeni | ≥40U/mg cholimba |
| DNase | Palibe chomwe chapezeka |
| RNase | Palibe chomwe chapezeka |
| Bioburden | ≤50CFU/g cholimba |
| Nucleic acid zotsalira | <5pg/mg olimba |
Katundu
| Gwero | Album ya Tritirachium |
| EC nambala | 3.4.21.64(Recombinant from Tritirachium album) |
| Kulemera kwa maselo | 29kDa (SDS-PAGE) |
| Isoelectric point | Chithunzi cha 7.81 |
| Mulingo wabwino kwambiri wa pH | 7.0-12.0 (Onse amachita ntchito zapamwamba) Fig.2 |
| Kutentha koyenera | 65℃ Chithunzi 3 |
| pH Kukhazikika | pH 4.5-12.5 (25℃,16h) Fig.4 |
| Kukhazikika kwamafuta | Pansi pa 50 ℃ (pH 8.0, 30min) Fig.5 |
| Kukhazikika kosungira | Kusungidwa pa 25 ℃ kwa 12months Fig.6 |
| Zoyambitsa | SDS, urea |
| Zoletsa | diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Zosungirako
Sungani ufa wa lyophilized pa -25~-15 ℃ kwa nthawi yayitali kutali ndi kuwala;Pambuyo pakutha, aliquot kukhala voliyumu yoyenera yosungirako kwakanthawi kochepa ku 2-8 ℃ kutali ndi kuwala kapena kusungidwa kwa nthawi yayitali pa -25 ~ -15 ℃ kutali ndi kuwala.
Kusamalitsa
Valani magalavu oteteza ndi magalasi pamene mukugwiritsa ntchito kapena poyeza sikelo, ndipo sungani mpweya wabwino mukaugwiritsa ntchito.Izi zitha kuyambitsa kusagwirizana ndi khungu komanso kuyabwa kwamaso.Ngati mutakokedwa, zingayambitse zizindikiro za chifuwa kapena mphumu kapena dyspnea.Zitha kuyambitsa kupsa mtima.
Tanthauzo la unit
Chigawo chimodzi cha NGS Protease K chimatanthauzidwa ngati kuchuluka kwa enzyme yofunikira kuti hydrolyze casein kukhala 1 μmol L-tyrosine pansi pa zikhalidwe zodziwika bwino.
Reagents kukonzekera
| Reagent | Wopanga | Catalog |
| Casein lusokuchokera ku mkaka wa ng'ombe | Sigma Aldrich | C7078 |
| NaOH | Sinopharm ChemicalMalingaliro a kampani Reagent Co., Ltd. | 10019762 |
| NaH2PO4· 2H2O | Sinopharm ChemicalMalingaliro a kampani Reagent Co., Ltd. | 20040718 |
| Na2HPO4 | Sinopharm ChemicalMalingaliro a kampani Reagent Co., Ltd. | 20040618 |
| Trichloroacetic acid | Sinopharm ChemicalMalingaliro a kampani Reagent Co., Ltd. | 80132618 |
| Sodium acetate | Sinopharm ChemicalMalingaliro a kampani Reagent Co., Ltd. | 10018818 |
| Acetic acid | Sinopharm ChemicalMalingaliro a kampani Reagent Co., Ltd. | 10000218 |
| HCl | Sinopharm ChemicalMalingaliro a kampani Reagent Co., Ltd. | 10011018 |
| Sodium carbonate | Sinopharm ChemicalMalingaliro a kampani Reagent Co., Ltd. | 10019260 |
| Foline-phenol | Sangon Biotech (Shanghai)Co., Ltd. | A500467-0100 |
| L-tyrosine | Sigma | 93829 |
Reagent I:
Gawo laling'ono: 1% Casein wochokera ku mkaka wa ng'ombe: Sungunulani 1g mkaka wa bovine casein mu 50ml wa 0.1M sodium phosphate solution, pH 8.0, kutentha mumadzi osamba pa 65-70 °C kwa mphindi 15, yambitsani ndi kupasuka, kuziziritsa ndi madzi, zosinthidwa ndi sodium hydroxide kuti pH 8.0, ndi kuchepetsa mu 100ml.
Reagent II:
Njira ya TCA: 0.1M trichloroacetic acid, 0.2M sodium acetate ndi 0.3M acetic acid (kulemera 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid motsatizana, kuwonjezera 50mL madzi opangidwa ndi deionized, sinthani ndi 4 dilute 3 pH, 4 dilute HCl ndi pH 3. 100 ml.)
Reagent III:
0.4m sodium carbonate solution (kulemera 4.24g anhydrous sodium carbonate ndi kupasuka mu 100mL madzi)
Reagent IV:
Folin phenol reagent: kuchepetsa nthawi 5 ndi madzi deionized.
Wothandizira V:
Enzyme diluent: 0.1 M sodium phosphate solution, pH 8.0.
Reagent VI:
L-tyrosine muyezo yothetsera: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml L-tyrosine kusungunuka ndi 0.2M HCl.
Ndondomeko
1. Yatsani UV-Vis spectrophotometer ndikusankha muyeso wa photometric.
2. Khazikitsani kutalika kwa mawonekedwe ngati 660nm.
3. Yatsani madzi osamba, ikani kutentha kwa 37 ℃, onetsetsani kutentha kosasintha kwa 3-5mins.
4. Preheat 0.5mL gawo lapansi mu chubu cha 2mL centrifuge pa 37 ℃ osamba madzi kwa 10mins.
5. Tulutsani njira ya 0.5mL ya enzyme mu preheated centrifuge chubu kwa mphindi 10.Khazikitsani enzyme diluent ngati gulu lopanda kanthu.
6. Onjezani 1.0 mL TCA reagent mwamsanga mutatha kuchita.Sakanizani bwino ndi kuukani mu osamba madzi kwa mphindi 30.
7. Centrifugate reaction solution.
8. Onjezani zigawo zotsatirazi mu dongosolo lomwe latchulidwa.
| Reagent | Voliyumu |
| Wopambana | 0.5 ml |
| 0.4M Sodium carbonate | 2.5 ml |
| Folin phenol reagent | 0.5 ml |
9. Sakanizani bwino musanayike m'madzi osamba 37 ℃ kwa mphindi 30.
10. OD660adadziwika ngati OD1;gulu lolamulira lopanda kanthu: Diluent ya enzyme imagwiritsidwa ntchito m'malo mwa enzyme njira yodziwira OD660ndi OD2, ΔOD=OD1-OD2.
11. L-tyrosine muyezo pamapindikira: 0.5mL osiyana ndende L-tyrosine njira, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent mu 5mL centrifuge chubu, incubate mu 37 ℃ kwa 30mins, zindikirani kwa OD660pamitundu yosiyanasiyana ya L-tyrosine, kenako adapeza njira yokhotakhota Y=kX+b, pomwe Y ndi kuchuluka kwa L-tyrosine, X ndi OD600.
Kuwerengera
2: Chiwerengero chonse cha yankho la mayankho (mL)
0.5: Kuchuluka kwa enzyme solution (mL)
0.5: Kuchuluka kwamadzimadzi komwe kumagwiritsidwa ntchito potsimikiza kwa chromogenic (mL)
10: Nthawi yochitirapo kanthu (mphindi)
Df: Dilution angapo
CKuchuluka kwa enzyme (mg/mL)
Ziwerengero
Fig.1 zotsalira za DNA
| Chitsanzo | Ave C4 | Nucleic Acid Kuchira (pg/mg) | Kuchira(%) | Total Nucleic Asidi ( p/mg) |
| Chithunzi cha PRK | 24.66 | 2.23 | 83% | 2.687 |
| PRK+STD2 | 18.723 | 126.728 | - | - |
| Mtengo wa STD1 | 12.955 |
- |
- |
- |
| Mtengo wa STD2 | 16 | |||
| Chithunzi cha STD3 | 19.125 | |||
| Mtengo wa STD4 | 23.135 | |||
| Chithunzi cha STD5 | 26.625 | |||
| RNA-Free H2O | Zosatsimikizika | - | - | - |
Fig. 2 Optimum pH
Mkuyu.3 Kutentha koyenera
Fig.4 pH Kukhazikika
Fig.5 Kukhazikika kwamafuta
Fig.6 Kukhazikika kosungira pa 25 ℃
