Pūtea K (Wai)
Cat Nama: HC4502A
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.Ka whakamahia nuitia mo tona whanuimotuhake.
Whakatakotoranga
| Te ahua | Kore tae ki te wai parauri marama |
| Mahi | ≥800 U/ml |
| Te kukū pūmua | ≥20 mg/ml |
| DNAase | Karekau i kitea |
| RNase | Karekau i kitea |
Nga Tikanga Rokiroki
Rokiroki i te pāmahana o te 2-8 ℃.
Āhuatanga
| nama EC | 3.4.21.64 (Rte whakakotahi mai i te pukapuka Tritirachium) |
| Te taumaha ngota | 29 kDa (SDS-WAHI) |
| Ikohikohiko | 7.81 |
| pH tino pai | 7.0-12.0 Fig.1 |
| Te pāmahana tino pai | 65 ℃ Fig.2 |
| pH pūmautanga | pH 4.5-12.5 (25 ℃, 16 h) Fig.3 |
| Te pūmautanga ngāwhā | Kei raro iho i te 50 ℃ (pH 8.0, 30 meneti) Fig.4 |
| 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 nga kano kano DNA 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 whakamatautau
Te whakamaramatanga o te waeine
Kotahi te waeine (U) kua tautuhia ko te nui o te enzyme e hiahiatia ana hei whakamaarama i te casein ki te whakaputa 1 μmol tyrosine 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 hydroxide ki te pH8.0, ka whakatika. rōrahi 100ml.
Reagent II: Te otinga TCA: 0.1M waikawa trichloroacetic, 0.2M konutai acetate, 0.3M waikawa acetate.
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: Te otinga Tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml tyrosine kua whakakorehia 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 i te 37 ℃ mo te 10mins.
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 i te 37 ℃ mo te 30mins.
5. OD660i whakatauhia hei OD1;rōpū whakahaere pātea: Ka whakamahia te 0.5ml reagent V hei whakakapi i te otinga whākōkī 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 Harbour Laboratory Press, Cold Spring Harbor(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 20mM K-phosphate buffer pH 8.0.Te kukū o te pūmua: 1mg/mL
Whakaahua 3 pH Te pumau
25 ℃, 16 h-maimoatanga me te otinga parepare 50mM: 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: 1mg/mL
Whakaahua 4 Ngawha pūmautanga
30 meneti-maimoatanga me te 50mM Tris-HCl buffer, pH 8.0.Te kukū o te pūmua: 1mg/mL
Fig. 5 Rokiroki pūmauty at 25℃
