Proteinase K NGS (ifu)
Injangwe No: HC4507A
NGS Protease K ni poroteyine ihamye ifite ibikorwa byinshi bya enzyme nibikorwa byihariye bya substrate. Enzyme ikunda kubora ester bond hamwe na peptide ihuza na C-terminal ya hydrophobique amino acide, sulfure irimo aside amine na acide aminide acide.Rero, ikoreshwa kenshi kugirango igabanye poroteyine muri peptide ngufi.NGS Protease K ni proteine isanzwe hamwe na Asp39-Yiwe69-Ser224inyabutatu ya catalitiki idasanzwe kuri proteine serine, hamwe na catalitiki ya centre ikikijwe na Ca Ca2+guhuza imbuga zo gutuza, gukomeza ibikorwa bya enzyme murwego rwo hejuru rwibihe.
Ibisobanuro
| Kugaragara | Ifu yera-yera-ifu ya amorphous, lyofilize |
| Igikorwa cyihariye | ≥40U / mg ikomeye |
| DNase | Nta n'umwe wamenyekanye |
| RNase | Nta n'umwe wamenyekanye |
| Bioburden | ≤50CFU / g ikomeye |
| Ibisigisigi bya aside nucleique | <5pg / mg ikomeye |
Ibyiza
| Inkomoko | Album ya Tritirachium |
| Numero ya EC | 3.4.21.64(Recombinant yo muri alubumu ya Tritirachium) |
| Uburemere bwa molekile | 29kDa (SDS-PAGE) |
| Ingingo ya Isoelectric | 7.81 Igishusho.1 |
| PH nziza | 7.0-12.0 (Bose bakora ibikorwa bihanitse) Igishusho.2 |
| Ubushyuhe bwiza | 65 ℃ Igishusho.3 |
| pH Guhagarara | pH 4.5-12.5 (25 ℃, 16h) Igishusho.4 |
| Ubushyuhe bukabije | Munsi ya 50 ℃ (pH 8.0, 30min) Igishusho.5 |
| Ububiko butajegajega | Yabitswe kuri 25 ℃ kumezi 12 Igishusho.6 |
| Abakangurambaga | SDS, urea |
| Inhibitor | Diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Ububiko
Bika ifu ya lyofilize kuri -25 ~ -15 ℃ igihe kinini kure yumucyo;Nyuma yo guseswa, aliquot mubunini bukwiye bwo kubika igihe gito kuri 2-8 ℃ kure yumucyo cyangwa ububiko bwigihe kirekire kuri -25 ~ -15 ℃ kure yumucyo.
Kwirinda
Wambare uturindantoki two kurinda hamwe na gogles mugihe ukoresha cyangwa upima, kandi ukomeze guhumeka neza nyuma yo kubikoresha.Iki gicuruzwa gishobora gutera uruhu rwa allergique no kurwara amaso.Niba ihumeka, irashobora gutera allergie cyangwa ibimenyetso bya asima cyangwa dyspnea.Birashobora gutera uburakari.
Igisobanuro cyibice
Igice kimwe cya NGS Protease K gisobanurwa nkubunini bwa enzyme isabwa kuri hydrolyze casein muri 1 μmol L-tyrosine mugihe cyagenwe gisanzwe.
Reagents kwitegura
| Reagent | Uruganda | Cataloge |
| Casein tekinikebiva mu mata y'inka | Sigma Aldrich | C7078 |
| NaOH | Sinopharm ChemicalReagent Co., Ltd. | 10019762 |
| NaH2PO4· 2H2O | Sinopharm ChemicalReagent Co., Ltd. | 20040718 |
| Na2HPO4 | Sinopharm ChemicalReagent Co., Ltd. | 20040618 |
| Acide Trichloroacetic | Sinopharm ChemicalReagent Co., Ltd. | 80132618 |
| Sodium acetate | Sinopharm ChemicalReagent Co., Ltd. | 10018818 |
| Acide acike | Sinopharm ChemicalReagent Co., Ltd. | 10000218 |
| HCl | Sinopharm ChemicalReagent Co., Ltd. | 10011018 |
| Sodium karubone | Sinopharm ChemicalReagent Co., Ltd. | 10019260 |
| Foline-fenol | Sangon Biotech (Shanghai)Co, Ltd. | A500467-0100 |
| L-tyrosine | Sigma | 93829 |
Reagent I:
Substrate: 1% Casein ivuye mumata ya bovine: gushonga 1g yamata ya bovine muri 50ml yumuti wa 0.1M sodium fosifate, pH 8.0, gushyushya ubwogero bwamazi kuri 65-70 ° C kuminota 15, kuvanga no gushonga, bikonje namazi, byahinduwe na sodium hydroxide kuri pH 8.0, hanyuma ukayungurura muri 100ml.
Reagent II:
Umuti wa TCA: 0.1M trichloroacetic aside, 0.2M sodium acetate na 0.3M acide acetike (ipima acide 1,64g trichloroacetic + 1.64g sodium acetate + 1.724mL acide acetike ikurikiranye, ongeramo amazi ya demioni 50mL, uhindure na HCl kuri pH 4.03, hanyuma uyunguruze 100ml).
Reagent III:
0.4m sodium ya karubone yumuti (ipima 4.24g ya karubone ya sodium ya anhydrous hanyuma igashonga mumazi 100mL)
Reagent IV:
Folin fenol reagent: kuyungurura inshuro 5 n'amazi ya deionion.
Reagent V:
Enzyme diluent: 0.1 M sodium fosifate yumuti, pH 8.0.
Reagent VI:
L-tyrosine igisubizo gisanzwe: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml L-tyrosine yashonga hamwe na 0.2M HCl.
Inzira
1. Fungura UV-Vis spectrophotometero hanyuma uhitemo gupima amafoto.
2. Shiraho uburebure bwa 660nm.
3. Fungura ubwogero bwamazi, shyira ubushyuhe kuri 37 ℃, urebe ko ubushyuhe budahinduka kuri 3-5mins.
4. Shyushya 0.5mL substrate muri 2mL ya centrifuge kuri 37 bath ubwogero bwamazi kuminota 10.
5. Kuramo 0.5mL ivanze na enzyme yumuti mumashanyarazi yashushe muminota 10.Shiraho enzyme diluent nkitsinda ryambaye ubusa.
6. Ongeramo 1.0 mL TCA reagent ako kanya nyuma yo kwitwara.Kuvanga neza hanyuma ugashyiramo ubwogero bwamazi muminota 30.
7. Centrifugate igisubizo cyibisubizo.
8. Ongeramo ibice bikurikira muburyo bwateganijwe.
| Reagent | Umubumbe |
| Indengakamere | 0.5 mL |
| 0.4M Sodium karubone | 2.5 mL |
| Folin reagent | 0.5 mL |
9. Kuvanga neza mbere yo gushiramo amazi yo koga 37 ℃ muminota 30.
10. OD660yagenwe nka OD1;itsinda rishinzwe kugenzura ubusa: Enzyme diluent ikoreshwa mugusimbuza enzyme igisubizo kugirango umenye OD660nka OD2, ΔOD = OD1-OD2.
11660kubintu bitandukanye bya L-tyrosine, hanyuma ibona umurongo usanzwe Y = kX + b, aho Y ni L-tyrosine yibanze, X ni OD600.
Kubara
2: Umubare wuzuye wibisubizo (mL)
0.5: Ingano yumuti wa enzyme (mL)
0.5: Igipimo cyamazi yakoreshejwe mugukoresha chromogenic (mL)
10: Igihe cyo kubyitwaramo (min)
Df: Gukoresha byinshi
C: Enzyme yibanze (mg / mL)
Imibare
Igicapo.1 ibisigisigi bya ADN
| Icyitegererezo | Ave C4 | Acide Nucleic Gukira (pg / mg) | Gukira (%) | Nucleic Acide ( 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-Yubusa H2O | Kutamenyekana | - | - | - |
Igishushanyo.2 pH nziza
Igishushanyo.3 Ubushyuhe bwiza
Igishushanyo.4 pH Guhagarara
Igicapo.5
Igishushanyo.6 Kubika neza kuri 25 ℃
