Proteinase K mNGS (amazi)
Proteinase K ni proteine ihamye hamwe na substrate yagutse.Itesha agaciro poroteyine nyinshi muri kavukire ndetse no imbere yimyenda.Ibimenyetso bivuye mubushakashatsi bwa kristu na molekuline byerekana ko enzyme ari iyumuryango wa subtilisin hamwe na site ikora catalitike triad (Asp39-Yiwe69-Ser224).Ikibanza cyiganjemo clavage ni peptide ihuza peptide yegeranye na carboxyl groupe ya aliphatic na aromatic amino acide hamwe na alpha amino yahagaritswe.Bikunze gukoreshwa muburyo bwagutse.Iyi proteinase K yagenewe umwihariko wa mNGS.Ugereranije nizindi poroteyine K, zirimo na aside nucleic nkeya yanduye hamwe nimikorere imwe ya enzymatique, ishobora kurushaho kwemeza mNGS kumanuka.
Ububiko
2-8 ℃ kumyaka 2
Ibisobanuro
| Kugaragara | Ibara ritagira ibara ryijimye |
| Igikorwa | 00800 U / ml |
| Intungamubiri za poroteyine | ≥20 mg / ml |
| Nickase | Nta n'umwe wamenyekanye |
| DNase | Nta n'umwe wamenyekanye |
| RNase | Nta n'umwe wamenyekanye |
Ibyiza
| Numero ya EC | 3.4.21.64(Recombinant yo muri alubumu ya Tritirachium) |
| Ingingo ya Isoelectric | 7.81 |
| PH nziza | 7.0- 12.0 Igishushanyo 1 |
| Ubushyuhe bwiza | 65 ℃ Ishusho 2 |
| pH ituze | pH 4.5- 12.5 (25 ℃, 16 h) Ishusho 3 |
| Ubushyuhe bukabije | Munsi ya 50 ℃ (pH 8.0, 30 min) Igishusho 4 |
| Ububiko butajegajega | Ibikorwa birenga 90% kumezi 12 kuri 25 ℃ |
| Abakangurambaga | SDS, urea |
| Inhibitor | Diisopropyl fluorophosphate;fenilmethylsulfonyl fluoride |
Porogaramu
1. Ibikoresho byo gusuzuma
2. Ibikoresho byo gukuramo RNA na ADN
3. Gukuramo ibice bitari poroteyine biva mu ngingo, kwangirika kwa poroteyine, nka ADNinkingo no gutegura heparin
4. Gutegura ADN ya chromosome na pulsed electrophorei
5. Iburengerazuba
6. Enzymatique glycosylated albumin reagents mugupima vitro
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 (U) gisobanurwa nkubunini bwa enzyme isabwa kuri hydrolyze casein kugirango itange 1 μ moltyrosine kumunota mubihe bikurikira.
Reagents kwitegura
Reagent I: 1g amata yamata yashonga muri 50ml yumuti wa 0.1M sodium fosifate (pH 8.0), yashizwemo mumazi 65-70 ℃ mumazi 15mins, akayungurura akayashonga, akonjeshwa namazi, agahindurwa na hydroxide ya sodium kuri pH 8.0, nubunini bugenwe. 100ml.
Reagent II: 0.1M acide trichloroacetic, 0.2M sodium acetate, 0.3M acide acetike.
Reagent III: 0.4M Na2CO3igisubizo.
Reagent IV: Forint reagent ivanze namazi meza inshuro 5.
Reagent V: Enzyme diluent: 0.1M sodium fosifate yumuti (pH 8.0).
Reagent VI: igisubizo cya tirozine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0,25 umol / ml tirozine yashonga hamwe na 0.2M HCl.
Inzira
1. 0.5ml ya reagent Nabanje gushyuha kugeza kuri 37 ℃, ongeramo 0.5ml yumuti wa enzyme, vanga neza, na incubate kuri37 ℃ kuminota 10.
2. Ongeramo 1ml ya reagent II kugirango uhagarike reaction, vanga neza, kandi ukomeze incubation kuri 30mins.
3. Centrifugate igisubizo cyibisubizo.
4. Fata 0.5ml ndengakamere, ongeramo 2.5ml reagent III, 0.5ml reagent IV, vanga neza hanyuma ube kuri 37 ℃kuri 30mins.
5. OD660yagenwe nka OD1;itsinda rishinzwe kugenzura ubusa: 0.5ml reagent V ikoreshwa mugusimbuza enzymeigisubizo cyo kumenya OD660nka OD2, ΔOD = OD1-OD2.
6660kubintu 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)
Reba
1. Wieger U & Hilz H. FEBS Mureke.(1972);23:77.
2. Wieger U & Hilz H. Biochem.Biophys.Res.Umuganda.(1971);44: 513.
3. Hilz, H.n'abandi.,Eur.J. Biochem.(1975);56: 103-108.
4. Sambrook J.et al., Gukwirakwiza Molecular: Igitabo cya Laboratoire, integuro ya 2, Icyambu gikonjeItangazamakuru rya Laboratoire, Icyambu gikonje (1989).
Imibare
Igishushanyo.1 Ibyiza pH
100mM igisubizo cya buffer: pH6.0-8.0, Na-fosifate;pH8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Ubunini bwa Enzyme: 1mg / mL
Igishushanyo.2 Ubushyuhe bwiza
Igisubizo muri 20 mM K-fosifate buffer pH 8.0.Enzyme yibanze: 1 mg / mL
Igishusho.3 pH Igihagararo
25 ℃, 16 h-kuvura hamwe na 50 mM igisubizo: pH 4.5- 5.5, Acetate;pH 6.0-8.0, Na-fosifate;pH 8.0-9.0, Tris-HCl.pH 9.0- 12.5, Glycine-NaOH.Enzyme yibanze: 1 mg / mL
Igishushanyo.4 ituze
30 min-kuvura hamwe na 50 mM Tris-HCl buffer, pH 8.0.Enzyme yibanze: 1 mg / mL
Ububiko.5 stability at 25 ℃
