Proteinase K (Ulwelo)
Inombolo yekati: HC4502A
I-Proteinase K yi-serine protease ezinzileyo ene-substrate ebanzi.Ithoba iiproteni ezininzi kwilizwe lasekuhlaleni naxa kukho izinto zokucoca.Ubungqina obuvela kwikristale kunye nezifundo zobume bemolekyuli bubonisa ukuba i-enzyme yeyosapho lwe-subtilisin enendawo esebenzayo ye-catalytic triad (Asp.39-Yakhe69- Iseva224).Eyona ndawo iphambili yokuqhekeka yi-peptide bond ekufuphi neqela le-carboxyl le-aliphatic kunye ne-amino acid enuka kamnandi enamaqela e-alpha amino avaliweyo.Ngokuqhelekileyo isetyenziselwa ngokubanziiinkcukacha ezithile.
Inkcazo
| Imbonakalo | Ulwelo olungenambala ukuya kumdaka okhanyayo |
| Umsebenzi | ≥800 U/ml |
| Ugxininiso lweprotheyini | ≥20 mg/ml |
| DNase | Akukho nanye ifunyenweyo |
| RNase | Akukho nanye ifunyenweyo |
Iimeko zokuGcina
Gcina kwiqondo lobushushu le-2-8℃.
Iipropati
| Inombolo yeEC | 3.4.21.64 (Riphuma kwicwecwe leTtirachium) |
| Ubunzima bemolekyuli | 29 kDa (SDS-PAGE) |
| Indawo ye-isoelectric | 7.81 |
| Eyona pH | 7.0-12.0 Umzobo.1 |
| Ubushushu obuphezulu | 65 ℃ Isazobe |
| ukuzinza kwe-pH | pH 4.5-12.5 (25℃, 16 h) Umfanekiso 3 |
| Ukuzinza kwe-Thermal | Ngaphantsi kwe-50℃ (pH 8.0, i-30 mins) Umfanekiso we-4 |
| Izivuseleli | SDS, urea |
| Izithinteli | I-Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Usetyenziso
1. Ikhithi yokuxilongwa kwemfuzo
2. I-RNA kunye ne-DNA extraction kits
3. Ukukhutshwa kwezinto ezingezizo iiprotheyini kwiithishu, ukuthotywa kokungcola kweeprotheni, ezifana nezitofu ze-DNA kunye nokulungiswa kwe-heparin.
4. Ukulungiswa kwe-chromosome DNA nge-pulsed electrophoresis
5. Iblothi yaseNtshona
6. I-enzymatic glycosylated albumin reagents in vitro diagnosis
Ukulumkela
Nxiba iiglavu zokhuseleko kunye neglavu xa usebenzisa okanye ulinganise ubunzima, kwaye ugcine umoya opholileyo emva kokusetyenziswa.Le mveliso inokubangela ukuchasana nesikhumba kunye nokucaphuka kakhulu kwamehlo.Ukuba uphefumle, kunokubangela i-allergies okanye iimpawu ze-asthma okanye i-dyspnea.Inokubangela ukucaphuka kokuphefumla.
Isivavanyi
Inkcazo yeyunithi
Iyunithi enye (U) ichazwa njengobungakanani be-enzyme efunekayo kwi-hydrolyze casein ukuvelisa i-1 μmol tyrosine ngomzuzu phantsi kweemeko ezilandelayo.
Ukulungiswa kwee-reagents
I-Reagent I: I-1g ye-casein yobisi yachithwa kwi-50ml ye-0.1M isisombululo se-sodium phosphate (pH 8.0), ifakwe kwi-65-70 ℃ yamanzi kwi-15mins, ixutywe kwaye ichithwe, ipholile ngamanzi, ilungelelaniswe yi-sodium hydroxide kwi-pH8.0, kwaye igxininisekile umthamo 100ml.
I-Reagent II: isisombululo se-TCA: i-0.1M i-trichloroacetic acid, i-0.2M i-acetate ye-sodium, i-0.3M i-acetic acid.
I-Reagent III: 0.4M Na2CO3isisombululo.
I-Reagent IV: I-Forint reagent ihlanjululwe ngamanzi acocekileyo ngamaxesha angama-5.
I-Reagent V: I-enzyme diluent: i-0.1M isisombululo se-sodium phosphate (pH 8.0).
I-Reagent VI: isisombululo seTyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml i-tyrosine echithwe kunye ne-0.2M HCl.
Inkqubo
1. I-0.5ml ye-reagent I ifudumala kwangaphambili ukuya kwi-37℃, yongeza i-0.5ml yesisombululo se-enzyme, xuba kakuhle, kwaye ufukamele kwi-37℃ nge-10mins.
2. Yongeza i-1ml ye-reagent II ukumisa ukusabela, udibanise kakuhle, kwaye uqhubeke nokufukamela i-30mins.
3. Isisombululo se-Centrifugate reaction.
4. Thatha i-0.5ml supernatant, yongeza i-2.5ml reagent III, i-0.5ml reagent IV, xuba kakuhle kwaye ufukamele kwi-37℃ i-30mins.
5. OD660kwamiselwa njenge OD1;iqela lokulawula elingenanto: I-0.5ml reagent V isetyenziselwa ukutshintsha isisombululo se-enzyme ukumisela i-OD660njengoko OD2, ΔOD=OD1-OD2.
6. I-L-tyrosine standard curve: 0.5mL i-concentration eyahlukileyo ye-L-tyrosine isisombululo, i-2.5mL Reagent III, i-0.5mL i-Reagent IV kwi-5mL centrifuge tube, incubate kwi-37℃ ye-30mins, ibone i-OD660yoxinaniso olwahlukileyo lwe-L-tyrosine, emva koko yafumana igophe eliqhelekileyo Y=kX+b, apho i-Y yi-concentration ye-L-tyrosine, i-X yi-OD600.
Ukubala
2: Umthamo opheleleyo wesisombululo sokusabela (mL)
0.5: Umthamo wesisombululo se-enzyme (mL)
0.5: Umthamo wolwelo we-reaction osetyenziswa kwi-chromogenic determination (mL)
10: Ixesha lokuphendula (imiz)
Df: Dilution ezininzi
CUxinzelelo lwe-enzyme (mg/mL)
Iimbekiselo
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.I-Biophys.Res.Uluntu.(1971);44:513.
3. Hilz, H.okqhubekayo.,I-eur.J. Biochem.(1975);56:103–108 .
4. USambrook Jet al., I-Molecular Cloning: Incwadi yeLabhoratri, i-2nd edition, i-Cold Spring Harbour Laboratory Press, i-Cold Spring Harbor (1989).
Amanani
ikhiwane. 1 Okona kulungileyo pH
Isisombululo se-buffer se-100mM: pH6.0-8.0, i-Na-phosphate;pH8.0- 9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Uxinzelelo lwe-Enzyme:1mg/mL
Umzobo 2 Owona bushushu bobushushu
Ukusabela kwi-20mM K-phosphate buffer pH 8.0.Uxinzelelo lwe-enzyme: 1mg/mL
Umzobo we-3 pH Uzinzo
25℃,16 h-unyango nge-50mM isisombululo se-buffer: pH 4.5-5.5, i-Acetate;pH 6.0-8.0, Na-phosphate;pH 8.0-9.0, Tris-I-HCl.pH 9.0-12.5, Glycine-NaOH.Uxinzelelo lwe-enzyme: 1mg/mL
Umzobo 4 Thermal uzinzo
Imizuzu engama-30 yonyango nge-50mM Tris-HCl buffer, pH 8.0.Uxinzelelo lwe-enzyme: 1mg/mL
Umzobo 5 Ugcino uzinzilety at 25℃
