Proteinase K NGS (Powder)
Cat No: HC4507A
NGS Protease K yog ib qho ruaj khov serine protease nrog kev ua haujlwm siab enzyme thiab dav substrate tshwj xeeb.Lub enzyme nyiam decomposes ester bonds thiab peptide bonds nyob ib sab ntawm C-terminal ntawm hydrophobic amino acids, sulfur-muaj amino acids thiab aromatic amino acids.Yog li, nws yog feem ntau siv los degrade proteins rau hauv luv peptides.NGS Protease K yog ib yam tshuaj serine protease nrog Asp39-Nws69-Ser224catalytic triad uas yog tshwj xeeb rau serine proteases, thiab lub catalytic chaw yog surrounded los ntawm cab Ca.2+khi qhov chaw rau stabilization, tswj kev ua haujlwm siab enzyme nyob rau hauv ntau yam kev mob.
Specification
| Qhov tshwm sim | Dawb mus rau dawb amorphous hmoov, lyophilized |
| Cov haujlwm tshwj xeeb | ≥40U / mg khoom |
| DNase | Tsis pom muaj leej twg |
| RNase | Tsis pom muaj leej twg |
| Bioburden | ≤50CFU / g khoom |
| Nucleic acid residue | <5pg/mg cov khoom |
Cov khoom
| Qhov chaw | Tritirachium album |
| EC tus lej | 3.4.21.64 Nws(Recombinant los ntawm Tritirachium album) |
| Molecular hnyav | 29kDa (SDS-PAGE) |
| Isoelectric point | 7.81 Ib |
| Qhov zoo tshaj pH | 7.0-12.0 (Txhua yam ua haujlwm siab) Fig.2 |
| Qhov kub zoo tshaj | 65 ℃ Fig.3 |
| pH stability | pH 4.5-12.5 (25 ℃, 16h) Fig.4 |
| Thermal stability | Hauv qab 50 ℃ (pH 8.0, 30min) Fig.5 |
| Cia ruaj khov | Khaws ntawm 25 ℃ rau 12 lub hlis Fig.6 |
| Activators | SDS, UA |
| Inhibitors | Diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Kev cia khoom
Khaws cov hmoov lyophilized ntawm -25 ~ -15 ℃ kom deb ntawm lub teeb;Tom qab dissolution, aliquot rau hauv ntim tsim nyog rau lub sij hawm luv luv cia ntawm 2-8 ℃ deb ntawm lub teeb los yog ntev cia ntawm -25 ~ -15 ℃ deb ntawm lub teeb.
Cov kev ceev faj
Hnav cov hnab looj tes tiv thaiv thiab tsom iav thaum siv los yog hnyav, thiab ua kom muaj cua zoo tom qab siv.Cov khoom no yuav ua rau cov tawv nqaij ua xua thiab mob qhov muag loj.Yog tias nqus tau, nws yuav ua rau muaj kev fab tshuaj lossis mob hawb pob lossis ua tsis taus pa.Tej zaum yuav ua rau ua pa ua pa.
Unit txhais
Ib chav tsev ntawm NGS Protease K yog txhais raws li tus nqi ntawm enzyme yuav tsum tau hydrolyze casein rau hauv 1 μmol L-tyrosine raws li tus qauv kev txiav txim.
Kev npaj reagent
| Reagent | Chaw tsim tshuaj paus | Catalog |
| Casein technicallos ntawm cov mis nyuj | Sigma Aldrich | c7078 ua |
| NaOH | Sinopharm tshuajReagent Co., Ltd. | 10019762 ib |
| NaH2PO4· 2 H2O | Sinopharm tshuajReagent Co., Ltd. | 20040718 |
| Na2HPO 4 | Sinopharm tshuajReagent Co., Ltd. | 20040618 |
| Trichloroacetic acid | Sinopharm tshuajReagent Co., Ltd. | 8 0132618 |
| Sodium acetate hmoov | Sinopharm tshuajReagent Co., Ltd. | 100 18818 |
| Acetic acid | Sinopharm tshuajReagent Co., Ltd. | 10000218 |
| HCl | Sinopharm tshuajReagent Co., Ltd. | 10011018 ib |
| Sodium carbonate | Sinopharm tshuajReagent Co., Ltd. | 100 19260 |
| Foline-phenol | Tuam Tshoj Biotechnology (Shanghai)Co., Ltd. | A500467-0100 |
| L-tyrosine | Sigma | 93829 ua |
Reagent kuv:
Substrate: 1% Casein los ntawm bovine mis nyuj tov: dissolve 1g bovine mis nyuj casein nyob rau hauv 50ml ntawm 0.1M sodium phosphate tov, pH 8.0, tshav kub hauv dej da dej ntawm 65-70 ° C rau 15mins, do thiab dissolve, txias nrog dej, kho los ntawm sodium hydroxide rau pH 8.0, thiab dilute rau 100ml.
Reagent II:
TCA tov: 0.1M trichloroacetic acid, 0.2M sodium acetate thiab 0.3M acetic acid (hnyav 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid successively, ntxiv 50mL deionized dej, kho nrog HCl rau 4.0, p. 100 ml).
Reagent III:
0.4m sodium carbonate tov (qhov hnyav 4.24g anhydrous sodium carbonate thiab yaj hauv 100mL dej)
Reagent IV:
Folin phenol reagent: dilute 5 zaug nrog dej deionized.
Reagent V:
Enzyme diluent: 0.1 M sodium phosphate kua, pH 8.0.
Reagent VI:
L-tyrosine txheem tov: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml L-tyrosine yaj nrog 0.2M HCl.
Txheej txheem
1. Qhib lub UV-Vis spectrophotometer thiab xaiv qhov ntsuas photometric.
2. Teem lub wavelength li 660nm.
3. Qhib dej da dej, teeb qhov kub thiab txias rau 37 ℃, xyuas kom qhov kub tsis hloov rau 3-5mins.
4. Preheat 0.5mL substrate nyob rau hauv 2mL centrifuge raj ntawm 37 ℃ dej da dej rau 10mins.
5. Extract 0.5mL diluted enzyme tov rau hauv lub preheated centrifuge raj rau 10 feeb.Teem lub enzyme diluent ua pab pawg dawb.
6. Ntxiv 1.0 mL TCA reagent tam sim ntawd tom qab cov tshuaj tiv thaiv.Sib tov zoo thiab incubate nyob rau hauv dej da dej rau 30 feeb.
7. Centrifugate cov tshuaj tiv thaiv.
8. Ntxiv cov khoom nram qab no hauv qhov kev txiav txim tau teev tseg.
| Reagent | Ntim |
| Supernatant | 0.5 ml yog ' |
| 0.4M Sodium carbonate | 2.5 ml ib |
| Folin phenol reagent | 0.5 ml yog ' |
9. Sib tov zoo ua ntej incubating hauv dej da dej 37 ℃ rau 30 mins.
10. OD660tau txiav txim raws li OD1;Pab pawg tswj hwm dawb paug: Enzyme diluent yog siv los hloov cov tshuaj enzyme los txiav txim OD660as OD2, OD = OD1- OD2.
11. L-tyrosine txheem nkhaus: 0.5mL txawv concentration L-tyrosine tov, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent nyob rau hauv 5mL centrifuge raj, incubate nyob rau hauv 37 ℃ rau 30mins, kuaj rau OD660rau qhov sib txawv ntawm L-tyrosine, ces tau tus qauv nkhaus Y = kX + b, qhov twg Y yog L-tyrosine concentration, X yog OD600.
Kev suav
2: Tag nrho cov ntim ntawm cov tshuaj tiv thaiv (mL)
0.5: Ntim ntawm enzyme tov (mL)
0.5: Cov tshuaj tiv thaiv kua ntim siv hauv kev txiav txim siab chromogenic (mL)
10: Lub sij hawm teb (min)
Df: Dilution ntau
CEnzyme concentration (mg / mL)
Cov duab
Fig.1 DNA residue
| Qauv | Ave C4 | Nucleic Acid Rov qab (pg / mg) | Rov qab (%) | Total Nucleic Acid ( pg / mg) |
| PRK | 24.66 Nws | 2.23 | 83% | 2.687 ib |
| PRK+STD2 | 18.723 ib | 126.728 ib | — | — |
| STD1 | 12.955 Nws |
— |
— |
— |
| STD2 | 16 | |||
| STD3 | 19.125 Nws | |||
| STD4 | 23.135 Nws | |||
| STD5 | 26.625 Nws | |||
| RNA-dawb H2O | Tsis paub meej | — | — | — |
Fig.2 Optimum pH
Fig.3 Qhov kub thiab txias
Fig.4 pH Stability
Fig.5 Thermal stability
Fig.6 Cia ruaj khov ntawm 25 ℃
