Proteinase K mNGS (zamadzimadzi)
Proteinase K ndi serine protease yokhazikika yokhala ndi gawo lalikulu la gawo lapansi.Imawononga mapuloteni ambiri m'dziko lakwawo ngakhale pamaso pa zotsukira.Umboni wochokera ku maphunziro a kristalo ndi mamolekyu akuwonetsa kuti enzymeyi ndi ya banja la subtilisin lomwe lili ndi malo othandizira othandizira atatu (Asp.39-Wake69-Ser224).Malo odziwika kwambiri a cleavage ndi chomangira cha peptide choyandikana ndi gulu la carboxyl la aliphatic ndi zonunkhira za amino acid okhala ndi magulu otsekedwa a alpha amino.Nthawi zambiri amagwiritsidwa ntchito pazambiri zake.proteinase K iyi idapangidwira mNGS mwapadera.Poyerekeza ndi proteinase K ina, imakhala ndi kuipitsidwa kwa nucleic acid yocheperako komanso magwiridwe antchito a enzymatic, omwe amatha kutsimikizira kugwiritsa ntchito kwa mNGS.
Zosungirako
2-8 ℃ kwa zaka 2
Kufotokozera
| Maonekedwe | Zamadzimadzi zopanda mtundu mpaka zofiirira |
| Zochita | ≥800 U/ml |
| Kukhazikika kwa Mapuloteni | ≥20 mg/ml |
| Nickase | Palibe chomwe chapezeka |
| DNase | Palibe chomwe chapezeka |
| RNase | Palibe chomwe chapezeka |
Katundu
| EC nambala | 3.4.21.64(Recombinant from Tritirachium album) |
| Isoelectric point | 7.81 |
| Mulingo wabwino kwambiri wa pH | 7.0-12.0 Chithunzi 1 |
| Kutentha koyenera | Chithunzi cha 65 ℃ |
| pH kukhazikika | pH 4.5- 12.5 (25 ℃, 16 h) Chithunzi 3 |
| Kukhazikika kwamafuta | Pansi pa 50 ℃ (pH 8.0, 30 min) Chithunzi 4 |
| Kukhazikika kosungira | Kupitilira 90% kwa miyezi 12 pa 25 ℃ |
| Zoyambitsa | SDS, urea |
| Zoletsa | diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Mapulogalamu
1. Zida zowunikira ma genetic
2. Zida zochotsera ma RNA ndi DNA
3. Kuchotsa zinthu zopanda puloteni kuchokera m’minyewa, kuwonongeka kwa zonyansa zamapuloteni, monga DNA.katemera ndi kukonzekera heparin
4. Kukonzekera kwa chromosome DNA ndi pulsed electrophoresis
5. Western blot
6. Enzymatic glycosylated albumin reagents in vitro diagnosis
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 (U) chimatanthauzidwa ngati kuchuluka kwa enzyme yofunikira kuti hydrolyze casein ipange 1 μmol.tyrosine pamphindi pazifukwa zotsatirazi.
Reagents kukonzekera
Reagent I: 1g mkaka casein anali kusungunuka mu 50ml wa 0.1M sodium mankwala njira (pH 8.0), chofungatira mu 65-70 ℃ madzi 15mins, analimbikitsa ndi kusungunuka, utakhazikika ndi madzi, kusinthidwa ndi sodium hydroxide kuti pH 8.0, ndi yokhazikika voliyumu. 100 ml.
Reagent II: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
Reagent III: 0.4M Na2CO3yankho.
Reagent IV: Forint reagent kuchepetsedwa ndi madzi oyera kwa 5 nthawi.
Reagent V: Enzyme diluent: 0.1M sodium phosphate solution (pH 8.0).
Reagent VI: yankho la tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine wosungunuka ndi 0.2M HCl.
Ndondomeko
1. 0.5ml ya reagent I imatenthedwa kale mpaka 37 ℃, onjezerani 0.5ml ya enzyme solution, sakanizani bwino, ndi kuumitsa pa.37 ℃ kwa mphindi 10.
2. Onjezerani 1ml ya reagent II kuti muyimitse zomwe zikuchitika, sakanizani bwino, ndikupitiriza kukulitsa kwa 30mins.
3. Centrifugate reaction solution.
4. Tengani 0.5ml supernatant, onjezerani 2.5ml reagent III, 0.5ml reagent IV, sakanizani bwino ndi kuumitsa pa 37 ℃kwa 30mins.
5. OD660adadziwika ngati OD1;gulu lopanda kanthu lowongolera: 0.5ml reagent V imagwiritsidwa ntchito m'malo mwa enzymenjira kudziwa OD660ndi OD2, ΔOD=OD1-OD2.
6. L-tyrosine muyezo pamapindikira: 0.5mL osiyana ndende L-tyrosine njira, 2.5mL Reagent III, 0.5mL Reagent IV mu 5mL centrifuge chubu, incubate mu 37 ℃ kwa 30mins, kudziwa 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)
Maumboni
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.Biophys.Res.Commun.(1971);44:513.
3. Ndi, H.ndi al.,EUR.J. Biochem.(1975);56:103–108 .
4. Sambrook Jet al., Kuphatikizika kwa mamolekyulu: Buku la Laboratory, kope lachiwiri, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989).
Ziwerengero
chith.1 Zabwino kwambiri pH
100mM yankho la bafa:pH6.0-8.0, Na-phosphate;pH8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Enzyme concentration:1mg/mL
Mkuyu.2 Kutentha koyenera
Zomwe zimachitika mu 20 mM K-phosphate buffer pH 8.0.Kuchuluka kwa enzyme: 1 mg/mL
Chithunzi cha 3 pH Kukhazikika
25 ℃, 16 h-mankhwala ndi 50 mM buffer yankho: 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.Kuchuluka kwa enzyme: 1 mg/mL
Chithunzi cha 4 Kutentha bata
30 min-mankhwala ndi 50 mM Tris-HCl buffer, pH 8.0.Kuchuluka kwa enzyme: 1 mg/mL
Fig.5 Kusungirako khazikikaty at 25 ℃
