Proteinase K mNGS (mokelikeli)
Proteinase K ke serine protease e tsitsitseng e nang le likarolo tse pharaletseng tsa substrate.E senya liprotheine tse ngata sebakeng sa matsoalloa esita le ka pel'a li-detergents.Bopaki bo tsoang lithutong tsa kristale le sebopeho sa limolek'hule bo bontša hore enzyme ke ea lelapa la subtilisin le nang le sebaka se sebetsang sa catalytic triad (Asp.39- Hae69-Ser224).Sebaka sa mantlha sa cleavage ke bond ea peptide e bapileng le sehlopha sa carboxyl sa li-amino acid tsa aliphatic le tse nkhang hamonate tse nang le lihlopha tsa alpha amino tse koetsoeng.Hangata e sebelisoa bakeng sa tlhaloso e pharaletseng ea eona.Proteinease K ena e etselitsoe mNGS ka ho khetheha.Ha e bapisoa le protheine K e 'ngoe, e na le tšilafalo e nyane ea nucleic acid e nang le ts'ebetso e ts'oanang ea enzymatic, e ka netefatsang ts'ebeliso e tlase ea mNGS.
Maemo a polokelo
2-8 ℃ bakeng sa lilemo tse 2
Tlhaloso
| Ponahalo | Seela se se nang mmala ho isa bosootho bo kganyang |
| Ketsahalo | ≥800 U/ml |
| Tšebeliso ea Liprotheine | ≥20 mg/ml |
| Nickase | Ha ho e fumanoeng |
| DNase | Ha ho e fumanoeng |
| RNase | Ha ho e fumanoeng |
Thepa
| Nomoro ea EC | 3.4.21.64(Recombinant ho tsoa ho albamo ea Tritirachium) |
| Ntlha ea Isoelectric | 7.81 |
| Optimum pH | 7.0- 12.0 Setšoantšo sa 1 |
| Optimum mocheso | 65 ℃ Setšoantšo sa 2 |
| pH botsitso | pH 4.5- 12.5 (25 ℃, 16 h) Setšoantšo sa 3 |
| Ho tsitsa ha mocheso | Ka tlase ho 50 ℃ (pH 8.0, 30 min) Setšoantšo sa 4 |
| Botsitso ba polokelo | Ho feta 90% ts'ebetso ea likhoeli tse 12 ho 25 ℃ |
| Li-activators | SDS, urea |
| Li-inhibitors | diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Lisebelisoa
1. Setsi sa tlhahlobo ea liphatsa tsa lefutso
2. RNA le DNA extraction kits
3. Ho ntšoa ha likaroloana tseo e seng tsa protheine ho tloha liseleng, ho senyeha ha litšila tsa protheine, joalo ka DNA.liente le ho lokisetsa heparin
4. Tokisetso ea chromosome DNA ka pulsed electrophoresis
5. Letlapa la Bophirimela
6. Enzymatic glycosylated albumin reagents in vitro diagnosis
Mehato ya tlhokomelo
Apara litlelafo le likhalase tsa tšireletso ha u sebelisa kapa u bekha boima ba 'mele, 'me u lule u e-na le moea o motle ka mor'a hore u li sebelise.Sehlahisoa sena se ka etsa hore letlalo le se ke la hana le ho teneha haholo.Haeba e hema, e ka baka matšoao a allergy kapa asthma kapa dyspnea.E ka baka ho teneha ha moea.
Tlhaloso ea yuniti
Yuniti e le 'ngoe (U) e hlalosoa e le palo ea enzyme e hlokahalang ho hydrolyze casein ho hlahisa 1 μmol.tyrosine ka motsotso tlas'a maemo a latelang.
Ho lokisoa ha li-reagents
Reagent I: 1g milk casein e ile ea qhibiliha ka 50ml ea 0.1M sodium phosphate solution (pH 8.0), e kenngoa ka 65-70 ℃ metsi bakeng sa 15mins, e tsosoa le ho qhibiliha, e pholile ka metsi, e lokisoa ke sodium hydroxide ho pH 8.0, le molumo o tsitsitseng o tsitsitseng 100ml.
Reagent II: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
Reagent III: 0.4M Na2CO3tharollo.
Reagent IV: Forint reagent e hlapolotsoe ka metsi a hloekileng ka makhetlo a 5.
Reagent V: Mokelikeli oa Enzyme: 0.1M tharollo ea sodium phosphate (pH 8.0).
Reagent VI: tharollo ea tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml tyrosine e qhibilihisitsoeng ka 0.2M HCl.
Tsamaiso
1. 0.5ml ea reagent I e futhumala pele ho 37℃, eketsa 0.5ml ea motsoako oa enzyme, kopanya hantle, 'me u e kenye ka37 ℃ bakeng sa metsotso e 10.
2. Eketsa 1ml ea reagent II ho emisa karabelo, kopanya hantle, 'me u tsoele pele ho kenya 30mins.
3. Centrifugate reaction solution.
4. Nka 0.5ml supernatant, eketsa 2.5ml reagent III, 0.5ml reagent IV, kopanya hantle 'me u etsetse ka 37℃.bakeng sa 30mins.
5. OD660e ile ea khethoa e le OD1;sehlopha sa taolo se se nang letho: 0.5ml reagent V e sebelisetsoa ho nkela enzyme sebakatharollo ea ho khetholla OD660joalo ka OD2, ΔOD=OD1-OD2.
6. Lekhalo le tloaelehileng la L-tyrosine: 0.5mL e fapaneng ea mahloriso L-tyrosine tharollo, 2.5mL Reagent III, 0.5mL Reagent IV ka 5mL centrifuge tube, incubate ka 37 ℃ bakeng sa 30mins, lemoha bakeng sa OD660bakeng sa mahloriso a fapaneng a L-tyrosine, joale a fumana lekhalo le tloaelehileng la Y=kX+b, moo Y e leng mahloriso a L-tyrosine, X ke OD600.
Palo
2: Kakaretso ea bolumo ea tharollo ea karabelo (mL)
0.5: Bophahamo ba tharollo ea enzyme (mL)
0.5: Bophahamo ba mokelikeli o sebetsang o sebelisitsoeng qetong ea chromogenic (mL)
10: Nako ea ho arabela (metsotso)
Df: Dilution multiple
CKhatiso ea enzyme (mg/mL)
Litšupiso
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.Lithuto tsa bophelo.Res.Komune.(1971);44:513.
3. Hilz, H.et al.,EUR.J. Biochem.(1975);56:103–108 .
4. Sambrook Jet al., Cloning ea Molek'hule: Buka ea Laeborari, khatiso ea 2, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989).
Lipalo
feie.1 Optimum pH
100mM tharollo ea buffer:pH6.0-8.0, Na-phosphate;pH8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Enzyme concentration:1mg/mL
Fig.2 Optimum mocheso
Maikutlo ho 20 mM K-phosphate buffer pH 8.0.Khatiso ea enzyme: 1 mg/mL
Setšoantšo sa 3 pH Botsitso
25 ℃, 16 h-phekolo e nang le tharollo ea 50 mM buffer: 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.Khatiso ea enzyme: 1 mg/mL
Setšoantšo sa 4 Thermal botsitso
Kalafo ea metsotso e 30 ka 50 mM Tris-HCl buffer, pH 8.0.Khatiso ea enzyme: 1 mg/mL
Setšoantšo sa 5 Storage botsitsoty at 25 ℃
