Proteinase K mNGS (wai)
Ko te Proteinase K he protease serine e mau ana me te tino whanui o te tïpako.Ka whakahekehia te maha o nga pūmua i roto i te ahua taketake ahakoa i te aroaro o nga kai horoi.Ko nga taunakitanga mai i nga rangahau o te hanga karaihe me te hanga rapoi e tohu ana no te whanau subtilisin te whaakiki me te triad catalytic pae kaha (Asp39-Tana69-Ser224).Ko te waahi nui o te wehenga ko te here peptide e tata ana ki te roopu carboxyl o nga waikawa amino aliphatic me te kakara me nga roopu alpha amino kua aukati.Kei te whakamahia nuitia mo tona whanui whanui.Ko tenei proteinase K he mea hanga motuhake mo te mNGS.Ka whakatauritea ki etahi atu proteinase K, he iti ake te poke o te waikawa karihi me te mahi enzymatic rite tonu, ka pai ake te tono mNGS ki raro.
Nga Tikanga Rokiroki
2-8 ℃ mo te 2 tau
Whakatakotoranga
| Te ahua | Kore tae ki te wai parauri marama |
| Mahi | ≥800 U/ml |
| Pūmua Pūmua | ≥20 mg/ml |
| Nickase | Karekau i kitea |
| DNAase | Karekau i kitea |
| RNase | Karekau i kitea |
Āhuatanga
| nama EC | 3.4.21.64(Te whakakotahi mai i te pukaemi Tritirachium) |
| Ikohikohiko | 7.81 |
| pH tino pai | 7.0- 12.0 Whakaahua 1 |
| Te pāmahana tino pai | 65 ℃ Whakaahua 2 |
| pH pūmautanga | pH 4.5- 12.5 (25 ℃, 16 h) Whakaahua 3 |
| Te pūmautanga ngāwhā | Kei raro iho i te 50 ℃ (pH 8.0, 30 min) Whakaahua 4 |
| Te pumau o te rokiroki | Neke atu i te 90% mahi mo te 12 marama i te 25 ℃ |
| Nga kaiwhakahohe | SDS, Urea |
| Nga kaitarai | Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Nga tono
1. Kete tātaritanga ira
2. Nga kete tangohanga RNA me te DNA
3. Te tangohanga o nga waahanga kore-poroti mai i nga kopa, te whakahekenga o nga poke o te pūmua, penei i te DNAkano kano me te whakarite heparin
4. Te whakarite i te DNA chromosome ma te hiko hiko
5. Te uru o te uru
6. Enzymatic glycosylated albumin reagents in vitro diagnostics
Whakatupato
Kia mau i nga karapu whakamarumaru me nga karaehe i te wa e whakamahi ana, e pauna ana ranei, kia pai te hau i muri i te whakamahinga.Ko tenei hua ka pa mai te mate mate kiri me te pawera kino o te kanohi.Mena ka ngohia, ka puta he mate mate mate, he tohu mate huango ranei, he kopa ranei.Ka raru pea te manawa.
Te whakamaramatanga o te waeine
Kotahi te waeine (U) kua tautuhia ko te nui o te whākōkī e hiahiatia ana hei whakamaroke i te casein kia puta ai te 1 μmoltyrosine ia meneti i raro i nga tikanga e whai ake nei.
Te whakarite reagents
Reagent I: 1g miraka casein i rewa i roto i te 50ml o te 0.1M konutai phosphate solution (pH 8.0), incubated i roto i te 65-70 ℃ wai mo te 15mins, whakaohokia me te rewa, whakamataohia e te wai, whakatikahia e te konutai waihā ki te pH 8.0, me te rōrahi pūmau. 100ml.
Reagent II: 0.1M waikawa trichloroacetic, 0.2M konutai acetate, 0.3M waikawa waikawa.
Reagent III: 0.4M Na2CO3otinga.
Reagent IV: Forint reagent kua diluted ki te wai parakore mo nga wa e 5.
Reagent V: Te waimeha enzyme: 0.1M wairewa konutai phosphate (pH 8.0).
Reagent VI: wairewa tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine kua memeha ki te 0.2M HCl.
Tikanga
1. Ko te 0.5ml o te reagent I kua whakamahana i mua ki te 37 ℃, tāpirihia te 0.5ml o te otinga whākōkī, uru pai, ka whakakororia ki37 ℃ mo te 10 meneti.
2. Taapirihia he 1ml o te reagent II kia mutu te tauhohenga, uru pai, ka haere tonu te heki mo te 30meneti.
3. Te otinga tauhohenga Centrifugate.
4. Tangohia te 0.5ml reagent, tāpirihia te 2.5ml reagent III, 0.5ml reagent IV, uru pai ka whakakororia ki te 37℃mo te 30 meneti.
5. OD660i whakatauhia hei OD1;rōpū whakahaere pātea: 0.5ml reagent V ka whakamahia hei whakakapi i te whākōkīotinga hei whakatau i te OD660hei OD2, ΔOD=OD1-OD2.
6. Anau paerewa L-tyrosine: 0.5mL rerekē te kukū L-tyrosine otinga, 2.5mL Reagent III, 0.5mL Reagent IV i roto i te 5mL centrifuge ngongo, incubate i roto i te 37℃ mo te 30mins, kitea mo te OD660mō te kukū rerekē o te L-tyrosine, kātahi ka whiwhi i te ānau paerewa Y=kX+b, ko Y te kukū L-tyrosine, X he OD600.
Tātaitanga
2: Tapeke rōrahi o te otinga tauhohenga (mL)
0.5: Rōrahi o te otinga whākōkī (mL)
0.5: Rōrahi wai tauhohenga e whakamahia ana ki te whakatau chromogenic (mL)
10: Wā tauhohenga (min)
Df: Te waimehatanga maha
C: Te kukū o te pūmua (mg/mL)
Tohutoro
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.Biophys.Res.Commun.(1971);44:513.
3. Hilz, H.et al.,Eur.J. Biochem.(1975);56:103–108.
4. Sambrook Jet al., Whakakotahitanga Molecular: He pukapuka taiwhanga, putanga tuarua, Cold Spring HarbourLaboratory Press, Cold Spring Harbour (1989).
Nga whika
Fig.1 Tino pai pH
100mM otinga parepare: pH6.0-8.0, Na-phosphate;pH8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Kutanga Enzyme:1mg/mL
Fig.2 Te pāmahana tino pai
Tauhohenga i roto i te 20 mM K-phosphate buffer pH 8.0.Te kukū o te pūmua: 1 mg/mL
Fig.3 pH Te pumau
25 ℃, 16 h-maimoatanga me te 50 mM otinga parepare: 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.Te kukū o te pūmua: 1 mg/mL
Fig.4 Ngawha pūmautanga
30 meneti-maimoatanga me te 50 mM Tris-HCl parepare, pH 8.0.Te kukū o te pūmua: 1 mg/mL
Fig.5 Rokiroki pūmauty at 25℃
