Proteinase K (Lyophilized pauta)
Numera pusi: HC4500A
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 lautelefa'apitoa.
Tulaga Teuteu
2-8 ℃ teuina mo taimi pupuu, -25 ~ -15 ℃ teuina mo taimi umi.Tulaga pa'u mamago -25~-15℃ aoga mo le 3 tausaga;e tatau ona fa'amavae le pa'u o le enzyme i le voluma talafeagai.
Fa'amatalaga
| Fa'aaliga | Pa'epa'e i le pa'epa'e amorphous lyophilized pauta |
| Gaoioiga | ≥30 U/mg |
| DNase | Leai se mea na iloa |
| RNase | Leai se mea na iloa |
Meatotino
| Numera EC | 3.4.21.64 (Recombinant mai le album Tritirachium) |
| mamafa mole | 29 kDa (SDS-PAGE) |
| 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℃, 16h) Ata.3 |
| Tulaga mafanafana | I lalo ole 50℃(pH 8.0, 30min) Ata.4 |
| 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 tui DNA ma sauniuniga ole heparin.
4. Sauniuniga o le chromosome DNA e ala i le pulsed 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.
Su'ega
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 μmol tyrosine 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: fa'afefeteina 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 faʻamavaeina i le 0.2M HCL.
Taualumaga
1. 0.5ml o le reagent I ua muamua faʻamafanafanaina i le 37 ℃, faʻaopopo le 0.5ml o le enzyme solution, faʻafefiloi lelei, ma faʻafefe i le 37 ℃ mo le 10mins.
2. Faaopopo le 1 ml o le reagent II e taofi ai le tali, faʻafefiloi lelei, ma faʻaauau le incubation 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 30mins.
5. OD660na fuafuaina o le OD1;vaega pulea avanoa: 0.5ml reagent V o loʻo faʻaaogaina e sui ai le enzyme solution e fuafua ai 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).
Ata 2 O le vevela sili ona lelei
Tali ile 20 mM K-phosphate buffer pH 8.0.Fa'asa'o ole Enzyme: 1mg/mL
Ata 3 pH Maumau
25 ℃, 16 h-togafitiga faʻatasi ma le 50mM 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: 1mg/mL
Ata 4 Tevela mautu
30 min-togafitiga ma le 50mM Tris-HCL paʻu, pH 8.0.Fa'asa'o ole Enzyme: 1mg/mL
Ata 5 Teuina stability at 25℃
