Proteinase K mNGS (suavai)
Proteinase K ose serine protease mautu ma lautele substrate specificity.E fa'aleagaina ai le tele o polotini i totonu o le atunu'u e o'o lava i le i ai o ni vaila'au.O faʻamaoniga mai suʻesuʻega tioata ma le molecular structure o loʻo faʻaalia ai o le enzyme e aʻafia i le subtilisin aiga ma se nofoaga galue catalytic triad (Asp).39-O lana69-Ser224).O le nofoaga autu o le cleavage o le sootaga peptide e lata ane i le vaega carboxyl o aliphatic ma aromatic amino acids ma poloka alpha amino vaega.E masani ona faʻaaogaina mo lona faʻapitoa lautele.O lenei proteinase K ua fa'apitoa mo le mNGS.Pe a faatusatusa i le isi proteinase K, o loʻo i ai le faʻaitiitia o le faʻaleagaina o le nucleic acid ma le faʻaogaina o le enzymatic tutusa, lea e sili atu ona mautinoa le faʻaogaina o le mNGS i lalo.
Tulaga Teuteu
2-8 ℃ mo le 2 tausaga
Fa'amatalaga
| Fa'aaliga | Le lanu enaena i le vai enaena |
| Gaoioiga | ≥800 U/ml |
| Fa'atosina Polotini | ≥20 mg/ml |
| Nickase | Leai se mea na iloa |
| DNase | Leai se mea na iloa |
| RNase | Leai se mea na iloa |
Meatotino
| Numera EC | 3.4.21.64(Recombinant mai le Tritirachium album) |
| Isoelectric point | 7.81 |
| pH sili ona lelei | 7.0- 12.0 Ata 1 |
| O le vevela sili ona lelei | 65 ℃ Ata 2 |
| pH mautu | pH 4.5-12.5 (25 ℃, 16 h) Ata 3 |
| Tulaga mafanafana | I lalo ole 50 ℃ (pH 8.0, 30 min) Ata 4 |
| Tulaga mautu | I luga ole 90% gaioiga mo le 12 masina ile 25 ℃ |
| Activators | SDS, urea |
| Fa'alavelave | Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Talosaga
1. Pusa su'esu'ega fa'a-genetic
2. RNA ma DNA faʻapipiʻi pusa
3. Aveesea o vaega e le o se polotini mai aano, faʻaleagaina o mea leaga ole polotini, e pei ole DNAtui ma sauniuniga o le heparin
4. Sauniuniga ole chromosome DNA e ala ile pulse electrophoresis
5. Western blot
6. Enzymatic glycosylated albumin reagents in vitro diagnostics
Fa'aeteetega
Fai totigilima ma mata tioata pe a fa'aaoga pe mamafa, ma fa'amama lelei pe a uma ona fa'aoga.O lenei oloa e ono fa'atupu ai le fa'afefe o le pa'u ma fa'aleagaina mata.Afai e manava, e mafai ona mafua ai le allergy po'o fa'ailoga sela po'o le manava.E ono mafua ai le manava manava.
Fa'amatalaga o le iunite
E tasi le iunite (U) o loʻo faʻamatalaina o le aofaʻi o le enzyme e manaʻomia e faʻafefe ai le casein e maua ai le 1 μmoltyrosine i le minute i lalo o tulaga nei.
Sauniuniga reagents
Reagent I: 1g susu casein na solo i le 50ml o le 0.1M sodium phosphate solution (pH 8.0), faʻapipiʻiina i le 65-70 ℃ vai mo le 15mins, faʻafefeteina ma faʻamavaeina, faʻamafanafanaina i le vai, fetuunai e le sodium hydroxide i le pH 8.0, ma faʻamautu voluma. 100ml.
Reagent II: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
Reagent III: 0.4M Na2CO3fofo.
Reagent IV: Forint reagent fa'afefe i le vai mama mo le 5 taimi.
Reagent V: Su'i fa'amamafa: 0.1M solution sodium phosphate (pH 8.0).
Reagent VI: vaifofo tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine fa'amavae ma le 0.2M HCl.
Taualumaga
1. 0.5ml o le reagent I ua muai mafanafana i le 37 ℃, faʻaopopo le 0.5ml o le enzyme solution, faʻafefiloi lelei, ma faʻafefe i le.37 ℃ mo le 10 minute.
2. Fa'aopoopo le 1ml o le reagent II e taofi ai le tali, fa'afefiloi lelei, ma fa'aauau le fa'asusu mo le 30mins.
3. Centrifugate fofo tali.
4. Ave le 0.5ml supernatant, faaopoopo 2.5ml reagent III, 0.5ml reagent IV, faʻafefiloi lelei ma faʻafefe i le 37 ℃mo le 30 minute.
5. OD660na fuafuaina o le OD1;vaega pulea avanoa: 0.5ml reagent V e faʻaaogaina e sui ai enzymefofo e iloa ai le OD660pei ole OD2, ΔOD=OD1-OD2.
6. L-tyrosine curve masani: 0.5mL eseese concentration L-tyrosine solution, 2.5mL Reagent III, 0.5mL Reagent IV i le 5mL centrifuge tube, incubate i le 37℃ mo 30mins, iloa mo OD660mo le fa'aogaina eseese o le L-tyrosine, ona maua lea o le fua fa'atatau Y=kX+b, lea Y o le L-tyrosine concentration, X o le OD.600.
Fa'atatauga
2: Aofa'i voluma o fofo tali (mL)
0.5: Volume ole vaifofo ole enzyme (mL)
0.5: Fa'aaogā le voluma vai fa'aaogā ile su'esu'ega chromogenic (mL)
10: Taimi e tali atu ai (min)
Df: Dilution tele
C: Fa'asa'o ole Enzyme (mg/mL)
Fa'asinomaga
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.Biophys.Re.Commun.(1971);44:513.
3. Hilz, H.et al.,Eur.J. Biochem.(1975);56:103–108.
4. Sambrook Jet al., Molecular Cloning: A Laboratory Manual, 2nd lomiga, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989).
Fa'atusa
Ata.1 Lelei pH
100mM fofo faʻafefe: pH6.0-8.0, Na-phosphate;pH8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Enzyme concentration:1mg/mL
Ata.2 O le vevela sili ona lelei
Tali ile 20 mM K-phosphate buffer pH 8.0.Fa'asa'o ole Enzyme: 1 mg/mL
Ata.3 pH Maumau
25 ℃, 16 h-togafitiga ma le 50 mM faʻafefe vaifofo: 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.Fa'asa'o ole Enzyme: 1 mg/mL
Ata.4 Tevela mautu
30 minute-togafitiga ma le 50 mM Tris-HCl paʻu, pH 8.0.Fa'asa'o ole Enzyme: 1 mg/mL
Ata.5 Teuina stability at 25℃
