Proteinase K mNGS (ulwelo)
I-Proteinase K yi-serine protease ezinzileyo ene-substrate ebanzi.Ithoba iiproteni ezininzi kwilizwe lasekuhlaleni naxa kukho izinto zokucoca.Ubungqina obuvela kwikristale kunye nezifundo zobume bemolekyuli bubonisa ukuba i-enzyme yeyosapho lwe-subtilisin enendawo esebenzayo ye-catalytic triad (Asp.39-Yakhe69-Ser224).Eyona ndawo iphambili yokuqhekeka yi-peptide bond ekufuphi neqela le-carboxyl le-aliphatic kunye ne-amino acid enuka kamnandi enamaqela e-alpha amino avaliweyo.Ngokuqhelekileyo isetyenziselwa ukucaciswa kwayo okubanzi.Le proteinase K yenzelwe ngokukodwa i-mNGS.Xa kuthelekiswa nenye iproteinase K, iqulethe ungcoliseko oluncinci lwe-nucleic acid kunye nokusebenza okufanayo kwe-enzymatic, enokuqinisekisa ngcono usetyenziso lwe-mNGS esezantsi.
Iimeko zokuGcina
2-8℃ iminyaka emi-2
Inkcazo
| Imbonakalo | Ulwelo olungenambala ukuya kumdaka okhanyayo |
| Umsebenzi | ≥800 U/ml |
| Iprotheyini yoxinaniso | ≥20 mg/ml |
| I-Nickase | Akukho nanye ifunyenweyo |
| DNase | Akukho nanye ifunyenweyo |
| RNase | Akukho nanye ifunyenweyo |
Iipropati
| Inombolo yeEC | 3.4.21.64(I-Recombinant evela kwi-albhamu ye-Ttirachium) |
| Indawo ye-isoelectric | 7.81 |
| Eyona pH | 7.0- 12.0 Umzobo 1 |
| Ubushushu obuphezulu | 65 ℃ Isazobe 2 |
| ukuzinza kwe-pH | pH 4.5- 12.5 (25 ℃, 16 h) Isazobe 3 |
| Ukuzinza kwe-Thermal | Ngaphantsi kwe-50 ℃ (pH 8.0, 30 min) Umzobo 4 |
| Uzinzo lokugcina | Ngaphezulu kwe-90% yomsebenzi weenyanga ezili-12 kuma-25 ℃ |
| Izivuseleli | SDS, urea |
| Izithinteli | I-Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Usetyenziso
1. Ikhithi yokuxilongwa kwemfuzo
2. I-RNA kunye ne-DNA extraction kits
3. Ukukhutshwa kwezinto ezingezizo iiprotheyini kwizicubu, ukuthotywa kokungcola kweeprotheni, njengeDNA.izitofu zokugonya kunye nokulungiswa kwe-heparin
4. Ukulungiswa kwe-chromosome DNA nge-pulsed electrophoresis
5. Iblothi yaseNtshona
6. I-enzymatic glycosylated albumin reagents in vitro diagnosis
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 (U) ichazwa njengobungakanani be-enzyme efunekayo kwi-hydrolyze casein ukuvelisa i-1 μmol.tyrosine ngomzuzu phantsi kwezi meko zilandelayo.
Ukulungiswa kwee-reagents
I-Reagent I: I-1g ye-casein yobisi yachithwa kwi-50ml ye-0.1M isisombululo se-sodium phosphate (pH 8.0), ifakwe kwi-65-70 ℃ yamanzi kwi-15mins, ixutywe kwaye ichithwe, ipholiswe ngamanzi, ilungiswe yi-sodium hydroxide kwi-pH 8.0, kunye nomthamo osisigxina. 100ml.
I-Reagent II: i-0.1M trichloroacetic acid, i-0.2M i-acetate ye-sodium, i-0.3M i-acetic acid.
I-Reagent III: 0.4M Na2CO3isisombululo.
I-Reagent IV: I-Forint reagent ihlanjululwe ngamanzi acocekileyo ngamaxesha angama-5.
I-Reagent V: I-enzyme diluent: i-0.1M isisombululo se-sodium phosphate (pH 8.0).
I-Reagent VI: isisombululo se-tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml i-tyrosine echithwa kunye ne-0.2M HCl.
Inkqubo
1. I-0.5ml ye-reagent I ifudunyezwe kwangaphambili ukuya kwi-37℃, yongeza i-0.5ml yesisombululo se-enzyme, xuba kakuhle, kwaye ufukamele37℃ imizuzu eyi-10.
2. Yongeza i-1ml ye-reagent II ukumisa ukusabela, udibanise kakuhle, kwaye uqhubeke nokufukamela i-30mins.
3. Isisombululo se-Centrifugate reaction.
4. Thatha i-0.5ml supernatant, yongeza i-2.5ml reagent III, 0.5ml reagent IV, xuba kakuhle kwaye ufukamele kwi-37℃imizuzu engama-30.
5. OD660kwamiselwa njenge OD1;iqela lokulawula elingenanto: I-0.5ml reagent V isetyenziselwa ukutshintsha i-enzymeisisombululo sokumisela i-OD660njengoko OD2, ΔOD=OD1-OD2.
6. I-L-tyrosine standard curve: 0.5mL i-concentration eyahlukileyo ye-L-tyrosine isisombululo, i-2.5mL Reagent III, i-0.5mL i-Reagent IV kwi-5mL centrifuge tube, incubate kwi-37℃ ye-30mins, ibone i-OD660yoxinaniso olwahlukileyo lwe-L-tyrosine, emva koko yafumana igophe eliqhelekileyo Y=kX+b, apho i-Y yi-concentration ye-L-tyrosine, 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)
Iimbekiselo
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.I-Biophys.Res.Uluntu.(1971);44:513.
3. Hilz, H.okqhubekayo.,I-eur.J. Biochem.(1975);56:103–108 .
4. USambrook Jet al., I-Molecular Cloning: Incwadi yeLabhoratri, i-2nd edition, i-Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989).
Amanani
ikhiwane.1 Okona kulungileyo pH
Isisombululo se-buffer se-100mM: pH6.0-8.0, i-Na-phosphate;pH8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Uxinzelelo lwe-Enzyme:1mg/mL
Umzobo.2 Olona bushushu buphezulu
Ukusabela kwi-20 mM K-phosphate buffer pH 8.0.Uxinzelelo lwe-enzyme: 1 mg / mL
Umzobo we-3 pH Uzinzo
I-25 ℃, i-16 h-unyango kunye ne-50 mM isisombululo se-buffer: pH 4.5- 5.5, i-Acetate;pH 6.0-8.0, Na-phosphate;pH 8.0-9.0, Tris-I-HCl.pH 9.0- 12.5, Glycine-NaOH.Uxinzelelo lwe-enzyme: 1 mg / mL
Umzobo.4 Thermal uzinzo
Imizuzu engama-30 yonyango nge-50 mM Tris-HCl buffer, pH 8.0.Uxinzelelo lwe-enzyme: 1 mg / mL
Fig.5 Ugcino uzinzilety at 25℃
