Proteinase K NGS (paura)
Cat Nama: HC4507A
Ko te NGS Protease K he protease serine e mau ana me te kaha o te mahi enzyme me te motuhake o te tïpako whanui. Ka pai te whakakore i nga here ester me nga here peptide e tata ana ki te C-terminal o nga waikawa amino hydrophobic, nga waikawa amino whanariki me nga waikawa amino kakara.Na, he maha nga wa e whakamahia ana ki te whakaheke i nga pūmua ki nga peptides poto.Ko NGS Protease K he protease serine angamaheni me te Asp39-Tana69-Ser224catalytic triad he mea ahurei ki te serine proteases, a ko te pokapū catalytic e karapotia ana e te tow Ca2+nga waahi here mo te whakapumautanga, te pupuri i te mahi nui o te enzyme i raro i te whānuitanga o nga tikanga.
Whakatakotoranga
| Te ahua | Te paura amorphous ma ki te ma-kore, kua lyophilized |
| Mahi motuhake | ≥40U/mg totoka |
| DNAase | Karekau i kitea |
| RNase | Karekau i kitea |
| Koiora | ≤50CFU/g totoka |
| Te toenga waikawa nucleic | <5pg/mg totoka |
Āhuatanga
| Puna | Pukaemi Tritirachium |
| nama EC | 3.4.21.64(Te whakakotahi mai i te pukaemi Tritirachium) |
| Te taumaha ngota | 29kDa (SDS-WAHI) |
| Ikohikohiko | 7.81 Fig.1 |
| pH tino pai | 7.0-12.0 (Katoa te mahi teitei) Fig.2 |
| Te pāmahana tino pai | 65℃ Fig.3 |
| pH Pūmautanga | pH 4.5-12.5 (25℃,16h) Fig.4 |
| Te pūmautanga ngāwhā | Kei raro iho i te 50 ℃ (pH 8.0, 30min) Fig.5 |
| Te pumau o te rokiroki | Ka penapena ki te 25 ℃ mo te 12marama Fig.6 |
| Nga kaiwhakahohe | SDS, Urea |
| Nga kaitarai | Diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Nga Tikanga Rokiroki
Whakaorangia te paura lyophilized i -25~-15 ℃ mo te wa roa atu i te marama;I muri i te whakakorenga, wehehia ki roto i te rōrahi e tika ana mo te rokiroki mo te wa poto i te 2-8 ℃ tawhiti atu i te marama me te rokiroki mo te wa roa i te -25 ~ -15 ℃ atu i te marama.
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
Ko tetahi waeine o NGS Protease K kua tautuhia ko te nui o te enzyme e hiahiatia ana hei whakamaarama i te casein ki te 1 μmol L-tyrosine i raro i nga tikanga whakatau paerewa.
Te whakarite reagents
| Reagent | Kaihanga | Putumōhio |
| Hangarau Caseinmai i te miraka kau | Sigma Aldrich | C7078 |
| NaOH | Sinopharm ChemicalReagent Co., Ltd. | 10019762 |
| NaH2PO4·2H2O | Sinopharm ChemicalReagent Co., Ltd. | 20040718 |
| Na2HPO4 | Sinopharm ChemicalReagent Co., Ltd. | 20040618 |
| Te waikawa trichloroacetic | Sinopharm ChemicalReagent Co., Ltd. | 80132618 |
| konutai acetate | Sinopharm ChemicalReagent Co., Ltd. | 10018818 |
| Waikawa acetic | Sinopharm ChemicalReagent Co., Ltd. | 10000218 |
| HCl | Sinopharm ChemicalReagent Co., Ltd. | 10011018 |
| konutai warowaihā | Sinopharm ChemicalReagent Co., Ltd. | 10019260 |
| Foline-phenol | Sangon Biotech (Shanghai)Co., Ltd. | A500467-0100 |
| L-tyrosine | Sigma | 93829 |
Reagent I:
Taapapa: 1% Casein mai i te waiu waiu bovine wairewa: whakakorikoria 1g bovine milk casein i roto i te 50ml o te 0.1M konutai phosphate solution, pH 8.0, whakamahana i roto i te pati wai i te 65-70 °C mo te 15mins, whakaohokia ka memeha, whakamatao ki te wai, whakatikahia e konutai waihā ki te pH 8.0, ka waimeha ki te 100ml.
Reagent II:
Te otinga TCA: 0.1M te waikawa trichloroacetic, 0.2M te konutai acetate me te 0.3M te waikawa acetic (te pauna 1.64g te waikawa trichloroacetic + 1.64g te konutai acetate + 1.724mL te waikawa acetate, ka taapirihia te 50mL wai whakakorehia, ka whakatika me te HCl3 ki te HCl3. 100ml).
Reagent III:
0.4m te wairewa konutai warowaiha (paunatia te 4.24g konutai waro warowaiwai, ka memeha ki te wai 100mL)
Reagent IV:
Folin phenol reagent: waimeha e 5 nga wa ki te wai whakaheke.
Reagent V:
Te waimeha pūmua: 0.1 M te wairewa konutai phosphate, pH 8.0.
Reagent VI:
L-tyrosine otinga paerewa: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml L-tyrosine rewa ki te 0.2M HCl.
Tikanga
1. Whakakāhia te UV-Vis spectrophotometer ka tohua te inenga whakaahua.
2. Tautuhia te roangaru hei 660nm.
3. Tahurihia te pati wai, tautuhia te mahana ki te 37 ℃, kia kore e rereke te pāmahana mo te 3-5mins.
4. Whakawerahia te tïpako 0.5mL i roto i te ngongo centrifuge 2mL i te 37 ℃ pati wai mo te 10 meneti.
5. Tangohia te 0.5mL wairewa whakaipoipo waimeha ki te ngongo centrifuge kua whakamahana mo te 10 meneti.Whakaritea te waimeha whākōkī hei rōpū pātea.
6. Tāpirihia te 1.0 mL TCA reagent i muri tonu i te tauhohenga.Whakaranu pai, ka whakamakuku ki roto i te pati wai mo te 30 meneti.
7. Te otinga tauhohenga Centrifugate.
8. Tāpirihia nga waahanga e whai ake nei i roto i te raupapa kua tohua.
| Reagent | Rōrahi |
| Hupiriana | 0.5 mL |
| 0.4M konutai warowaihā | 2.5 mL |
| Folin phenol reagent | 0.5 mL |
9. Whakaranu pai i mua i te whakamau ki roto i te pati wai he 37 ℃ mo te 30 meneti.
10. OD660i whakatauhia hei OD1;rōpū whakahaere pātea: Ka whakamahia te diluent enzyme hei whakakapi i te otinga whākōkī hei whakatau i te OD660hei OD2, ΔOD=OD1-OD2.
11. Anau paerewa L-tyrosine: 0.5mL rerekē te kukū L-tyrosine otinga, 2.5mL 0.4M konutai warowaihā, 0.5mL Folin phenol reagent i roto i te 5mL centrifuge ngongo, incubate i roto i te 37 ℃ mo te 30mins, kitea mo te OD660mo te kuhanga rereke o te L-tyrosine, katahi ka whiwhi i te anau paerewa Y=kX+b, ko Y te kuhanga 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)
Nga whika
Fig.1 te toenga DNA
| Tauira | Ave C4 | Waikawa Nucleic Whakaora(pg/mg) | Whakaora(%) | Nucleic Tapeke Waikawa ( wh/mg) |
| PRK | 24.66 | 2.23 | 83% | 2.687 |
| PRK+STD2 | 18.723 | 126.728 | — | — |
| STD1 | 12.955 |
— |
— |
— |
| STD2 | 16 | |||
| STD3 | 19.125 | |||
| STD4 | 23.135 | |||
| STD5 | 26.625 | |||
| RNA-Kore H2O | Kare i whakatau | — | — | — |
Fig.2 pH tino pai
Fig.3 Te pāmahana tino pai
Fig.4 pH Pūmautanga
Fig.5 Pumau te werawera
Fig.6 Pūmautanga rokiroki i te 25 ℃
