Proteinase K mNGS (kua kua)
Proteinase K yog ib qho ruaj khov serine protease nrog dav substrate tshwj xeeb.Nws degrades ntau proteins nyob rau hauv ib txwm lub xeev txawm nyob rau hauv lub xub ntiag ntawm detergents.Cov ntaub ntawv pov thawj los ntawm cov kev tshawb fawb siv lead ua thiab cov qauv molecular qhia tias cov enzyme belongs rau tsev neeg subtilisin nrog rau qhov chaw ua haujlwm catalytic triad (Asp.39-Nws69-Ser224).Qhov chaw tseem ceeb ntawm cleavage yog peptide daim ntawv cog lus nyob ib sab ntawm pawg carboxyl ntawm aliphatic thiab aromatic amino acids nrog thaiv alpha amino pawg.Nws yog feem ntau siv rau nws qhov dav dav.Cov proteinase K no tshwj xeeb tsim los rau mNGS.Piv nrog rau lwm cov proteinase K, nws muaj txawm tias tsawg dua nucleic acid paug nrog tib lub enzymatic kev ua tau zoo, uas tuaj yeem ua kom zoo dua rau hauv daim ntawv thov mNGS.
Kev cia khoom
2-8 ℃ rau 2 xyoos
Specification
| Qhov tshwm sim | Colorless rau lub teeb xim av kua |
| Kev ua si | ≥800 U / ml |
| Protein concentration | ≥ 20 mg / ml |
| Nickase | Tsis pom muaj leej twg |
| DNase | Tsis pom muaj leej twg |
| RNase | Tsis pom muaj leej twg |
Cov khoom
| EC tus lej | 3.4.21.64 Nws(Recombinant los ntawm Tritirachium album) |
| Isoelectric point | 7.81 ib |
| Qhov zoo tshaj pH | 7.0-12.0 Ib |
| Qhov kub zoo tshaj | 65 ℃ Daim duab 2 |
| pH stability | pH 4.5-12.5 (25 ℃, 16 h) Daim duab 3 |
| Thermal stability | Hauv qab 50 ℃ (pH 8.0, 30 min) Daim duab 4 |
| Cia ruaj khov | Tshaj 90% kev ua haujlwm rau 12 lub hlis ntawm 25 ℃ |
| Activators | SDS, UA |
| Inhibitors | Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Daim ntawv thov
1. Cov khoom siv kuaj kab mob caj ces
2. RNA thiab DNA rho cov khoom siv
3. Extraction tsis-protein Cheebtsam ntawm cov ntaub so ntswg, degradation ntawm protein impurities, xws li DNAtshuaj tiv thaiv thiab kev npaj ntawm heparin
4. Kev npaj ntawm chromosome DNA los ntawm pulsed electrophoresis
5. Western blot
6. Enzymatic glycosylated albumin reagents hauv vitro kuaj mob
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 (U) txhais tau tias yog tus nqi ntawm enzyme yuav tsum tau hydrolyze casein los tsim 1 μmol.tyrosine ib feeb nyob rau hauv cov xwm txheej hauv qab no.
Kev npaj reagent
Reagent I: 1g mis nyuj casein tau yaj hauv 50ml ntawm 0.1M sodium phosphate tov (pH 8.0), incubated nyob rau hauv 65-70 ℃ dej rau 15mins, stirred thiab yaj, txias los ntawm dej, kho los ntawm sodium hydroxide rau pH 8.0, thiab tsau ntim. 100 ml.
Reagent II: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
Reagent III: 0.4M Na2CO3daws.
Reagent IV: Forint reagent diluted nrog dej ntshiab rau 5 zaug.
Reagent V: Enzyme diluent: 0.1M sodium phosphate tov (pH 8.0).
Reagent VI: tyrosine daws: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml tyrosine yaj nrog 0.2M HCl.
Txheej txheem
1. 0.5ml ntawm reagent Kuv yog pre-warmed rau 37 ℃, ntxiv 0.5ml ntawm enzyme tov, sib tov zoo, thiab incubate ntawm37 ℃ rau 10 mins.
2. Ntxiv 1ml ntawm reagent II kom nres cov tshuaj tiv thaiv, sib tov zoo, thiab txuas ntxiv incubation rau 30mins.
3. Centrifugate cov tshuaj tiv thaiv.
4. Noj 0.5ml supernatant, ntxiv 2.5ml reagent III, 0.5ml reagent IV, sib tov zoo thiab incubate ntawm 37 ℃rau 30 mins.
5. OD660tau txiav txim raws li OD1;Pab pawg tswj hwm dawb paug: 0.5ml reagent V yog siv los hloov enzymedaws los txiav txim OD660as OD2, OD = OD1- OD2.
6. L-tyrosine txheem nkhaus: 0.5mL txawv concentration L-tyrosine tov, 2.5mL Reagent III, 0.5mL Reagent IV 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 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 ntaub ntawv
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77 ib.
2. Wieger U & Hilz H. Biochem.Biophys.Res.Pawg.(1971);44:51 3.
3. Hlo, H.thiab al.,Eur.J. Biochem.(1975);56:103–108.
4. Sambrook Jet al., Molecular Cloning: Phau Ntawv Qhia Kev Tshawb Fawb, 2nd ed, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989).
Cov duab
Fig.1 Qhov zoo pH
100mM tsis haum tshuaj: pH6.0-8.0, Na-phosphate;pH 8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Enzyme concentration: 1mg/mL
Fig.2 Qhov kub thiab txias
Cov tshuaj tiv thaiv hauv 20 mM K-phosphate tsis pH 8.0.Enzyme concentration: 1 mg / mL
fig.3 pH Kev ruaj ntseg
25 ℃, 16 h-kev kho mob nrog 50 mM buffer tov: 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.Enzyme concentration: 1 mg / mL
Fig.4 Thermal ruaj khov
30 min-kev kho mob nrog 50 mM Tris-HCl tsis, pH 8.0.Enzyme concentration: 1 mg / mL
Fig.5 Cia ruaj khovty at 25 ℃
