Proteinase K NGS (umgubo)
Inombolo yekati: HC4507A
I-NGS Protease K yi-serine protease ezinzileyo kunye nomsebenzi ophezulu we-enzyme kunye ne-substrate ebanzi.Ke, ihlala isetyenziselwa ukuthoba iiproteni zibe ziipeptide ezimfutshane.I-NGS Protease K yi-serine protease eqhelekileyo kunye ne-Asp39-Yakhe69-Ser224i-catalytic triad ekhethekileyo kwi-serine proteases, kwaye iziko le-catalytic lijikelezwe yi-tow Ca.2+iindawo zokubopha ukuzinzisa, ukugcina umsebenzi ophezulu we-enzyme phantsi koluhlu olubanzi lweemeko.
Inkcazo
| Imbonakalo | Emhlophe ukuya emhlophe emhlophe amorphous powder, lyophilized |
| Umsebenzi othile | ≥40U/mg eqinileyo |
| DNase | Akukho nanye ifunyenweyo |
| RNase | Akukho nanye ifunyenweyo |
| Umthwalo wezinto eziphilayo | ≤50CFU / g eqinileyo |
| Intsalela ye-Nucleic acid | <5pg/mg eqinileyo |
Iipropati
| Umthombo | I-albhamu ye-Ttirachium |
| Inombolo yeEC | 3.4.21.64(I-Recombinant evela kwi-albhamu ye-Ttirachium) |
| Ubunzima bemolekyuli | 29kDa (SDS-PAGE) |
| Indawo ye-isoelectric | 7.81 Isazobe |
| Eyona pH | 7.0-12.0 (Bonke benza umsebenzi ophezulu) Fig.2 |
| Ubushushu obuphezulu | 65℃ Isazobe.3 |
| pH Uzinzo | pH 4.5-12.5 (25℃,16h) Umfanekiso we-4 |
| Ukuzinza kwe-Thermal | Ngaphantsi kwe-50 ℃ (pH 8.0, 30min) Umzobo |
| Uzinzo lokugcina | Igcinwe kwi-25℃ ye-12months Fig.6 |
| Izivuseleli | SDS, urea |
| Izithinteli | I-Diisopropyl fluorophosphate;i-benzylsulfonyl fluoride |
Iimeko zokuGcina
Gcina i powder lyophilized kwi -25 ~ -15 ℃ ixesha elide kude nokukhanya;Emva kokuchithwa, i-aliquot ibe ngumthamo ofanelekileyo wokugcinwa kwexesha elifutshane kwi-2-8 ℃ kude nokukhanya okanye ukugcinwa kwexesha elide kwi -25 ~ -15 ℃ kude nokukhanya.
Ukulumkela
Nxiba iiglavu zokhuseleko kunye neglavu xa usebenzisa okanye ulinganise ubunzima, kwaye ugcine umoya opholileyo emva kokusetyenziswa.Le mveliso inokubangela ukuchasana nesikhumba kunye nokucaphuka kakhulu kwamehlo.Ukuba uphefumle, kunokubangela i-allergies okanye iimpawu ze-asthma okanye i-dyspnea.Inokubangela ukucaphuka kokuphefumla.
Inkcazo yeyunithi
Iyunithi enye ye-NGS iProtease K ichazwa njengobungakanani be-enzyme efunekayo kwi-hydrolyze casein kwi-1 μmol L-tyrosine phantsi kweemeko zokumisela umgangatho.
Ukulungiswa kwee-reagents
| I-Reagent | Umenzi | Ikhathalogu |
| Casein technicalukusuka kubisi lwenkomo | Sigma Aldrich | C7078 |
| NAOH | Sinopharm ChemicalI-Reagent Co., Ltd. | 10019762 |
| NaH2PO4·2H2O | Sinopharm ChemicalI-Reagent Co., Ltd. | 20040718 |
| Na2HPO4 | Sinopharm ChemicalI-Reagent Co., Ltd. | 20040618 |
| I-Trichloroacetic acid | Sinopharm ChemicalI-Reagent Co., Ltd. | 80132618 |
| I-acetate yesodium | Sinopharm ChemicalI-Reagent Co., Ltd. | 10018818 |
| Acetic acid | Sinopharm ChemicalI-Reagent Co., Ltd. | 10000218 |
| I-HCl | Sinopharm ChemicalI-Reagent Co., Ltd. | 10011018 |
| I-sodium carbonate | Sinopharm ChemicalI-Reagent Co., Ltd. | 10019260 |
| I-Foline-phenol | I-Sangon Biotech (eShanghai)Co., Ltd. | A500467-0100 |
| L-tyrosine | Sigma | 93829 |
I-Reagent I:
I-Substrate: 1% I-Casein evela kwisisombululo sobisi lwenkomo: nyibilikisa i-1g ye-bovine milk casein kwi-50ml ye-0.1M isisombululo se-sodium phosphate, i-pH 8.0, ubushushu kwibhafu yamanzi kwi-65-70 °C nge-15mins, xuba kwaye unyibilike, uphole ngamanzi, ulungelelanise i-sodium hydroxide ukuya kwi-pH 8.0, kwaye ihlambulule kwi-100ml.
I-Reagent II:
Isisombululo se-TCA: 0.1M i-trichloroacetic acid, i-0.2M i-acetate ye-sodium kunye ne-0.3M i-acetic acid (ubunzima be-1.64g trichloroacetic acid + 1.64g i-acetate ye-sodium + 1.724mL i-acetic acid ngokulandelanayo, yongeza i-50mL yamanzi e-deionized, ukulungelelanisa ne-4. 100ml).
I-Reagent III:
I-0.4m isisombululo se-sodium carbonate (ubunzima be-4.24g i-anhydrous sodium carbonate kwaye ichithe kwi-100mL yamanzi)
I-Reagent IV:
I-Folin phenol reagent: nciphisa amaxesha ama-5 ngamanzi adibeneyo.
Isixhobo V:
I-enzyme diluent: 0.1 M isisombululo se-sodium phosphate, i-pH 8.0.
I-Reagent VI:
Isisombululo esiqhelekileyo se-L-tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml L-tyrosine echithwe kunye ne-0.2M HCl.
Inkqubo
1. Vula i-UV-Vis spectrophotometer kwaye ukhethe umlinganiselo we-photometric.
2. Seta ubude bobude njenge-660nm.
3. Vula ukuhlamba kwamanzi, setha ubushushu kwi-37 ℃, qinisekisa ukuba ukushisa akutshintshi kwi-3-5mins.
4. Hlaziya i-substrate ye-0.5mL kwi-tube ye-2mL ye-centrifuge kwi-37℃ yokuhlamba amanzi kwi-10mins.
5. Khipha i-0.5mL isisombululo se-enzyme ehlanjululweyo kwi-tube ye-centrifuge yangaphambi kwe-10 imizuzu.Seta i-enzayimu yokuxutywa njengeqela elingenanto.
6. Yongeza i-1.0 mL TCA reagent ngokukhawuleza emva kokusabela.Xuba kakuhle kwaye ufukame kwindawo yokuhlambela amanzi imizuzu engama-30.
7. Isisombululo se-Centrifugate reaction.
8. Yongeza la macandelo alandelayo ngolandelelwano oluchaziweyo.
| I-Reagent | Umthamo |
| Onamandla | 0.5 ml |
| I-0.4M yeSodium carbonate | 2.5 ml |
| I-Folin phenol reagent | 0.5 ml |
9. Xuba kakuhle phambi kokufukamela kwibhafu yamanzi nge-37℃ imizuzu engama-30.
10. OD660kwamiselwa njenge OD1;iqela elingenanto yokulawula: I-enzyme diluent isetyenziselwa ukubuyisela isisombululo se-enzyme ukumisela i-OD660njengoko OD2, ΔOD=OD1-OD2.
11. I-L-tyrosine standard curve: 0.5mL i-concentration eyahlukileyo ye-L-tyrosine isisombululo, i-2.5mL 0.4M i-Sodium carbonate, i-0.5mL i-Folin phenol reagent kwi-5mL centrifuge tube, incubate kwi-37℃ kwi-30mins, fumana i-OD660yoxinaniso olwahlukileyo lwe-L-tyrosine, emva koko yafumana igophe elisemgangathweni Y=kX+b, apho uY yi-L-tyrosine yoxinaniso, i-X yi-OD600.
Ukubala
2: Umthamo opheleleyo wesisombululo sokusabela (mL)
0.5: Umthamo wesisombululo se-enzyme (mL)
0.5: Umthamo wolwelo we-reaction osetyenziswa kwi-chromogenic determination (mL)
10: Ixesha lokuphendula (imiz)
Df: Dilution ezininzi
CUxinzelelo lwe-enzyme (mg/mL)
Amanani
Umzobo.1 intsalela yeDNA
| Isampulu | Ave C4 | I-Nucleic Acid Ukubuyisela kwakhona(pg/mg) | Ubuyiselo(%) | Iyonke Nucleic Iasidi ( p/mg) |
| I-PRK | 24.66 | 2.23 | 83% | 2.687 |
| I-PRK+STD2 | 18.723 | 126.728 | - | - |
| I-STD1 | 12.955 |
- |
- |
- |
| I-STD2 | 16 | |||
| I-STD3 | 19.125 | |||
| I-STD4 | 23.135 | |||
| I-STD5 | 26.625 | |||
| RNA-Free H2O | Ukungamiselwanga | - | - | - |
Fig.2 Eyona pH ilungileyo
Umzobo.3 Olona bushushu buphezulu
Fig.4 pH Ukuzinza
Umzobo.5 Ukuzinza kwe-Thermal
Umzobo.6 Uzinzo lokugcina kwi-25℃
