Proteinase K mNGS (dareere)
Proteinase K waa serine protease deggan oo leh substrate gaar ah.Waxay hoos u dhigtaa borotiinno badan oo ku jira gobolka hooyo xitaa marka ay joogaan saabuunta.Caddaynta laga helay daraasadaha qaab dhismeedka kristal iyo molecular waxay tilmaamaysaa insaymku ay iska leeyihiin qoyska subtilisin oo leh goob firfircoon oo firfircoon (Asp).39-Kiisa69- Ser224).Goobta ugu badan ee jeexjeexa ayaa ah isku-xidhka peptide ee ku dheggan kooxda karboxyl ee asiidhyada amino ee aliphatic iyo caraf udgoon oo leh kooxo alfa amino ah oo xannibmay.Waxaa caadi ahaan loo isticmaalaa gaar ahaan ballaaran.Proteinase K waxaa si gaar ah loogu talagalay mNGS.Marka la barbar dhigo borotiinka K kale, waxa ku jira wasakhaynta aashitada nucleic yar oo leh isla waxqabadka enzymatic, kaas oo si fiican u hubin kara codsiga mNGS-ka hoose.
Xaaladaha Kaydinta
2-8 ℃ ilaa 2 sano
Tilmaamid
| Muuqashada | Midab la'aan iyo dareere bunni khafiif ah |
| Hawsha | ≥800 U/ml |
| Isku-ururinta borotiinka | ≥20 mg/ml |
| Nickase | Midna lama ogaan |
| DNA | Midna lama ogaan |
| RNase | Midna lama ogaan |
Guryaha
| lambarka EC | 3.4.21.64(Recombinant from Tritirachium album) |
| Barta koronto | 7.81 |
| pH ugu fiican | 7.0- 12.0 Jaantuska 1 |
| Heerkulka ugu wanaagsan | 65 ℃ Jaantuska 2 |
| xasilloonida pH | pH 4.5- 12.5 (25 ℃, 16 h) Jaantuska 3 |
| Deganaanshaha kulaylka | Ka hooseeya 50 ℃ (pH 8.0, 30 min) Jaantuska 4 |
| xasiloonida kaydinta | In ka badan 90% dhaqdhaqaaqa 12 bilood at 25 ℃ |
| Dhaqdhaqaaqayaasha | SDS, urea |
| Kahortagayaasha | Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Codsiyada
1. Xirmada ogaanshaha hidda-socodka
2. Qalabka soo saarista RNA iyo DNA
3. Ka soo saarida qaybaha aan borotiinka ahayn ee unugyada, hoos u dhaca wasakhda borotiinka, sida DNA-datallaalada iyo diyaarinta heparin
4. Diyaarinta koromosoomyada DNA-da oo la garaaco electrophoresis
5. Reer galbeed
6. Enzymatic glycosylated albumin reagents in vitro ogaanshaha
Ka taxadaritaan
Xidho galoofyada ilaalinta iyo muraayadaha marka aad isticmaalayso ama miisaanayso, si fiicanna hawo u sii isticmaal ka dib.Alaabtani waxay keeni kartaa fal-celin xasaasiyad maqaarka ah iyo cuncun daran oo indhaha ah.Haddii la nuugo, waxay keeni kartaa xasaasiyad ama calaamadaha neefta ama dyspnea.Waxa laga yaabaa inay keento cuncun xagga neefsiga ah.
Qeexitaanka unugga
Hal unug (U) ayaa lagu qeexaa sida cadadka insaymka looga baahan yahay in lagu shubo casein si loo soo saaro 1 μmoltyrosine daqiiqadiiba xaaladaha soo socda.
Reagents diyaarinta
Reagent I: 1g caanaha casein waxaa lagu milmay 50ml oo ah 0.1M sodium phosphate solution (pH 8.0), oo lagu shubay 65-70 ℃ biyo 15mins, walaaqay oo la milmay, biyo lagu qaboojiyey, lagu hagaajiyay sodium hydroxide ilaa pH 8.0, iyo mugga go'an 100ml.
Reagent II: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
Reagent III: 0.4M Na2CO3xal.
Reagent IV: Reagent Forint waxaa lagu qasi jiray biyo saafi ah ilaa 5 jeer.
Reagent V: Diluent Enzyme: 0.1M sodium phosphate solution (pH 8.0).
Reagent VI: Xalka tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine oo lagu milmay 0.2M HCl.
Habraaca
1. 0.5ml of reagent I waa hore loo diiray ilaa 37 ℃, ku dar 0.5ml oo ah xal enzyme, si fiican isku qas, oo ku shubDubo 37 ℃ 10 daqiiqo.
2. Ku dar 1ml oo reagent II ah si aad u joojiso fal-celinta, si fiican isku qas, oo sii wad soo-saarka 30mins.
3. Xalka falcelinta centrifugate.
4. Qaado 0.5ml supernatant, ku dar 2.5ml reagent III, 0.5ml reagent IV, si fiican isku walaaq oo ku dhaji 37℃30 daqiiqo.
5. OD660waxaa loo go'aamiyay sida OD1;Kooxda xakamaynta bannaan: 0.5ml reagent V waxaa loo isticmaalaa in lagu beddelo ensaymkaxal si loo go'aamiyo OD660sida OD2, ΔOD=OD1-OD2.
6. Qallooca caadiga ah ee L-tyrosine: 0.5mL kala duwanaansho fiirsashada L-tyrosine, 2.5mL Reagent III, 0.5mL Reagent IV ee tube centrifuge 5mL, ku dhufo 37 ℃ 30mins, ogow OD660fiirsashada kala duwan ee L-tyrosine, ka dibna la helay qalooca caadiga ah Y=kX+b, halkaas oo Y ay tahay xoojinta L-tyrosine, X waa OD600.
Xisaabinta
2: Wadarta guud ee xalalka falcelinta (ml)
0.5: Mugga xalinta enzyme (mL)
0.5: Mugga dareeraha falcelinta loo isticmaalo go'aaminta chromogenic (mL)
10: Waqtiga falcelinta (min)
Df: Kala-saar badan
CXakamaynta enzyme (mg/mL)
Tixraacyo
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.BiophysRes.Bulsho(1971);44:513.
3. Hilz, H.iyo al.,YuuroJ. Biochem(1975);56:103-108.
4. Sambrook Jet al., Cloning Molecular: Buugga Shaybaadhka, daabacaadda 2aad, Harbor Spring ColdSaxaafadda Shaybaadhka, Cold Spring Harbor (1989).
Sawirro
Sawirka.1 ugu fican pH
Xalka 100mM: pH6.0-8.0, Na-phosphate;pH8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Enzyme-fiirsashada: 1mg/mL
Jaantuska.2 Heerkulka ugu fiican
Falcelinta ku jirta 20 mM K-fosfate buffer pH 8.0.Heerarka Enzyme: 1 mg/mL
Jaantuska.3 pH Xasilooni
25 ℃, 16 h-daawaynta oo leh 50 mM xal kaydinta: 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.Heerarka Enzyme: 1 mg/mL
Jaantus.4 Kulaylka xasiloonida
30 daqiiqo oo daweyn ah oo leh 50mM Tris-HCl kaydiye, pH 8.0.Heerarka Enzyme: 1 mg/mL
Jaantus.5 Kaydinta xasilinty at 25℃
