Proteinase K (Lyophilized powder)
Katsi Nhamba: HC4500A
Proteinase K yakagadzikana serine protease ine yakafara substrate chaiyo.Iyo inosvibisa mapuroteni akawanda munharaunda yekuzvarwa kunyange pamberi pezvidziviriro.Humbowo kubva kukristaro uye mamorekuru magadzirirwo zvidzidzo zvinoratidza kuti enzyme ndeye subtilisin mhuri ine inoshanda saiti catalytic triad (Asp.39-Zvake69- Ser224)Nzvimbo huru ye cleavage ndiyo peptide bond iri padyo necarboxyl boka realiphatic uye anonhuwirira amino acids ane akavharika alpha amino mapoka.Inowanzoshandiswa kune yakafara yayospecificity.
Storage Conditions
2-8 ℃ kuchengetwa kwenguva pfupi, -25 ~ -15 ℃ kuchengetwa kwenguva refu.Dry poda mamiriro -25 ~ -15 ℃ inoshanda kwemakore matatu;iyo enzyme poda inofanira kunyungudutswa muhuwandu hwakakodzera.
Tsanangudzo
| Chitarisiko | Ichena kusvika kune-chena amorphous lyophilized powder |
| Chiitiko | ≥30 U/mg |
| DNase | Hapana chaonekwa |
| RNase | Hapana chaonekwa |
Properties
| EC nhamba | 3.4.21.64 (Recombinant from Tritirachium album) |
| Molecular uremu | 29 kDa (SDS-PEJI) |
| Isoelectric point | 7.81 |
| Optimum pH | 7.0-12.0 Fig.1 |
| Optimum tembiricha | 65 ℃ Fig.2 |
| pH kugadzikana | pH 4.5-12.5 (25℃, 16h) Fig.3 |
| Thermal kugadzikana | Below 50℃(pH 8.0, 30min) Fig.4 |
| Activators | SDS, urea |
| Inhibitors | diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Applications
1. Genetic diagnostic kit
2. RNA uye DNA extraction kits
3. Kubviswa kwezvinhu zvisiri zveprotein kubva munyama, kuora kweprotein tsvina, zvakadai seDNA vaccines uye kugadzirira heparin.
4. Kugadzirira kwechromosome DNA ne pulsed electrophoresis
5. Western blot
6. Enzymatic glycosylated albumin reagents in vitro diagnosis
Zvekungwaririra
Pfeka magirovhosi uye magirazi ekudzivirira paunenge uchishandisa kana kuyera, uye gara uine mhepo yakanaka mushure mekushandisa.Ichi chigadzirwa chinogona kukonzera ganda allergic reaction uye zvakanyanya kushatirwa kwemaziso.Kana inhaled, inogona kukonzera allergies kana asthma zviratidzo kana dyspnea.Inogona kukonzera kushatirwa kwekufema.
Assay
Tsanangudzo yeyuniti
Imwe unit (U) inotsanangurwa sehuwandu hwe enzyme inodiwa hydrolyze casein kugadzira 1 μmol tyrosine paminiti pasi pemamiriro anotevera.
Reagents kugadzirira
Reagent I: 1g mukaka casein yakanyungudutswa mu 50ml ye 0.1M sodium phosphate solution (pH 8.0), yakaiswa mu 65-70 ℃ mvura kwemaminetsi gumi nemashanu, yakamutswa uye yakanyungudutswa, yakanyoroveswa nemvura, inogadziriswa ne sodium hydroxide kusvika pH 8.0, uye yakagadziriswa vhoriyamu. 100ml.
Reagent II: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
Reagent III: 0.4M Na2CO3mhinduro.
Reagent IV: Forint reagent yakanyungudutswa nemvura yakachena ka5.
Reagent V: enzyme diluent: 0.1M sodium phosphate solution (pH 8.0).
Reagent VI: tyrosine mhinduro: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine yakanyungudutswa ne0.2M HCL.
Maitiro
1. 0.5ml ye reagent I inofanodziya kusvika ku37℃, wedzera 0.5ml ye enzyme solution, sanganisa zvakanaka, uye incubate pa 37℃ kwe 10mins.
2. Wedzera 1 ml ye reagent II kuti umise maitiro, sanganisa zvakanaka, uye ramba uchifukidzira kwe30mins.
3. Centrifugate reaction solution.
4. Tora 0.5ml supernatant, wedzera 2.5ml reagent III, 0.5ml reagent IV, sanganisa zvakanaka uye incuba pa 37℃ kwema30mins.
5. OD660yakatemwa seOD1;blank control group: 0.5ml reagent V inoshandiswa kutsiva enzyme mhinduro kuona OD660seOD2, DOD=OD1-OD2.
6. L-tyrosine yakajairika curve: 0.5mL yakasiyana-siyana L tyrosine solution, 2.5mL Reagent III, 0.5mL Reagent IV mu 5mL centrifuge tube, incubate mu 37 ℃ kwe30mins, tsvaga OD660nokuda kwekusangana kwakasiyana kweL-tyrosine, ndokubva yawana yakajairwa curve Y=kX+b, apo Y iri L- tyrosine concentration, X iri OD.600.
Calculation
2: Yese vhoriyamu yemhinduro mhinduro (mL)
0.5: Vhoriyamu ye enzyme solution (mL)
0.5: Reaction fluid volume inoshandiswa mukutsunga kwechromogenic (mL)
10: Nguva yekuita (min)
Df: Dilution yakawanda
CKuwedzera kwe enzyme (mg/mL)
References
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.Biophys.Res.Commun.(1971);44:513.
3. Hilz, H.uye al.,Eur.J. Biochem.(1975);56:103–108.
4. Sambrook Jet al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989).
Fig. 2 Optimum tembiricha
Reaction mu 20 mM K-phosphate buffer pH 8.0.Enzyme concentration: 1mg/mL
Fig. 3 pH Kugadzikana
25 ℃, 16 h-kurapa ne 50mM buffer mhinduro: 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: 1mg/mL
Mufananidzo 4 Thermal kugadzikana
30 min-kurapwa ne50mM Tris-HCL buffer, pH 8.0.Enzyme concentration: 1mg/mL
Fig. 5 Kuchengeta stability at 25℃
