I-Proteinase K (i-Lyophilized powder) HC4500A Isithombe Esifakiwe

I-proteinase K (impuphu ye-Lyophilized)

Inombolo yekati: HC4500A

Iphakheji: 100mg/1g/10g/100g/500g

Mahhala DNase, RNase, Nickase

Umsebenzi: ≥30 U/mg

Impilo yeshelufu iminyaka emi-3

Ezokuthutha ekamelweni lokushisa

Umthamo weqoqo elilodwa 30kg

Thumela Uphenyo

Incazelo Yomkhiqizo

Imininingwane yomkhiqizo

Idatha

Inombolo yekati: HC4500A

I-Proteinase K iyi-serine protease ezinzile enokucaciswa okubanzi kwe-substrate.Yehlisa isithunzi samaprotheni amaningi endaweni yendabuko ngisho nalapho kukhona izinto zokuhlanza.Ubufakazi obuvela ocwaningweni lwe-crystal nesakhiwo samangqamuzana bukhombisa ukuthi i-enzyme ingeyomndeni we-subtilisin ene-site catalytic triad esebenzayo (Asp₃₉-Wakhe₆₉- Iseva₂₂₄).Indawo eyinhloko yokuqhekeka isibopho se-peptide esiseduze neqembu le-carboxyl lama-amino acid aliphatic kanye nephunga elimnandi elinamaqembu e-alpha amino avinjiwe.Ivame ukusetshenziselwa ububanzi bayoukucacisa.

Okwedlule: Iphrotheni K NGS (powder) HC4507A

Olandelayo: 2×PCR Super Mix (eneDye) HCR2012A

Izimo Zokugcina

2-8℃ isitoreji sesikhathi esifushane,-25~-15℃ isitoreji sesikhathi eside.Isimo sempushana esomile -25~-15℃ sisebenza iminyaka emi-3;i-enzyme powder kufanele ichithwe ibe umthamo ofanele.

Ukucaciswa

Ukubukeka	Impushana emhlophe ukuya kokumhlophe e-amorphous lyophilized
Umsebenzi	≥30 U/mg
DNase	Akukho okutholiwe
RNase	Akukho okutholiwe

Izakhiwo

Inombolo ye-EC	3.4.21.64 (I-Recombinant evela ku-albhamu ye-Ttirachium)
Isisindo samangqamuzana	29 kDa (SDS-PAGE)
Iphuzu le-isoelectric	7.81
I-pH engcono kakhulu	7.0-12.0 Fig.1
Izinga lokushisa eliphezulu	65 ℃ Fig.2
pH ukuzinza	pH 4.5-12.5 (25℃, 16h) Fig.3
Ukuzinza okushisayo	Ngaphansi kuka-50℃(pH 8.0, 30min) Fig.4
Izishoshovu	SDS, urea
Ama-inhibitors	I-Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride

Izinhlelo zokusebenza

1. Ikhithi yokuxilonga yofuzo

2. I-RNA kanye ne-DNA extraction kits

3. Ukukhishwa kwezingxenye ezingezona amaprotheni ezicutshini, ukuwohloka kokungcola kwamaprotheni, njengemithi yokugomela i-DNA kanye nokulungiswa kwe-heparin

4. Ukulungiswa kwe-chromosome DNA nge-pulsed electrophoresis

5. Ibala laseNtshonalanga

6. Ama-enzymatic glycosylated albumin reagent in vitro diagnostic

Izinyathelo zokuzivikela

Gqoka amagilavu okuzivikela kanye nezibuko lapho usebenzisa noma ukala, futhi uhlale unomoya omuhle ngemva kokuwusebenzisa.Lo mkhiqizo ungase ubangele ukungezwani nesikhumba kanye nokucasuka okukhulu kwamehlo.Uma uhogele, kungase kubangele ukungezwani komzimba noma izimpawu zesifuba somoya noma i-dyspnea.Ingabangela ukucasuka kokuphefumula.

Isivivinyo

Incazelo yeyunithi

Iyunithi eyodwa (U) ichazwa njengenani le-enzyme edingekayo ukuze i-hydrolyze i-casein ukukhiqiza i-1 μmol tyrosine ngomzuzu ngaphansi kwezimo ezilandelayo.

Ukulungiswa kwama-reagents

I-Reagent I: I-1g yobisi i-casein yahlakazwa ku-50ml we-0.1M yesisombululo se-sodium phosphate (pH 8.0), ifakwe emanzini angu-65-70 ℃ imizuzu engu-15, inyakaziswa futhi incibilike, ipholiswe ngamanzi, ilungiswa nge-sodium hydroxide ibe yi-pH 8.0, kanye nevolumu ehleliwe 100ml.

I-Reagent II: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.

I-Reagent III: 0.4M Na₂CO₃isisombululo.

I-Reagent IV: I-Forint reagent ihlanjululwe ngamanzi ahlanzekile izikhathi ezi-5.

I-Reagent V: i-enzyme diluent: 0.1M isisombululo se-sodium phosphate (pH 8.0).

I-Reagent VI: isixazululo se-tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml i-tyrosine ehlakazwe ngo-0.2M HCL.

Inqubo

1. I-0.5ml ye-reagent I ifudunyezwa ngaphambilini ibe ngu-37℃, engeza u-0.5ml wesisombululo se-enzyme, uhlanganise kahle, futhi ufukamele ku-37℃ imizuzu engu-10.

2. Engeza i-1 ml ye-reagent II ukuze umise ukusabela, uhlanganise kahle, futhi uqhubeke nokufukamela imizuzu engu-30.

3. Isixazululo se-Centrifugate reaction.

4. Thatha i-0.5ml supernatant, engeza u-2.5ml reagent III, 0.5ml reagent IV, uhlanganise kahle futhi ufukamele ku-37℃ imizuzu engu-30.

5. OD₆₆₀kwanqunywa njenge-OD₁;iqembu lokulawula elingenalutho: I-0.5ml reagent V isetshenziselwa ukufaka isisombululo se-enzyme ukuze kunqunywe i-OD₆₆₀njengoba OD₂, ΔOD=OD₁-OD₂.

6. Ijika elijwayelekile le-L-tyrosine: 0.5mL isixazululo se-concentration esihlukile se-L tyrosine, 2.5mL Reagent III, 0.5mL Reagent IV ku-5mL centrifuge tube, incubate ku-37℃ imizuzu engu-30, thola i-OD₆₆₀ekuhlanganiseni okuhlukile kwe-L-tyrosine, kwase kutholakala ijika elijwayelekile elingu-Y=kX+b, lapho u-Y ewugxilisano lwe-L- tyrosine, u-X engu-OD₆₀₀.

Ukubala

2: Isamba sevolumu yesixazululo sokusabela (mL)

0.5: Umthamo wesixazululo se-enzyme (mL)

0.5: Ivolumu yoketshezi yokusabela esetshenziswa ekunqumeni kwe-chromogenic (mL)

10: Isikhathi sokuphendula (imizuzu)

Df: Dilution multiple

CUkugxila kwe-enzyme (mg/mL)

Izikhombo

1. Wieger U & Hilz H. FEBS Lett.(1972); 23:77.

2. Wieger U & Hilz H. Biochem.I-Biophys.I-Res.Umphakathi.(1971); 44:513.

3. Hilz, H.et al.,I-Eur.J. Biochem.(1975);56:103–108.

4. Sambrook Jet al., I-Molecular Cloning: Incwadi Yelabhorethri, uhlelo lwesi-2, i-Cold Spring HarborLaboratory Press, i-Cold Spring Harbor (1989).

Amanani

Fig. 1Okungcono kakhulu pH

Isixazululo sebhafa esingu-100mM: pH6.0-8.0, Na-phosphate;pH8.0-9.0, Tris-HCL;pH9.0-12.5, Glycine-NaOH.Enzyme concentration:1mg/mL

Umdwebo 2 Izinga lokushisa eliphezulu

Ukusabela ku-20 mM K-phosphate buffer pH 8.0.Ukuhlushwa kwe-enzyme: 1mg/mL

Umdwebo 3 pH Ukuzinza

25℃, 16 h-ukwelashwa ngesixazululo sebhafa esingu-50mM: 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.Ukuhlushwa kwe-enzyme: 1mg/mL

Umdwebo 4 Thermal ukuzinza

30 amaminithi wokwelapha nge-50mM Tris-HCL buffer, pH 8.0.Ukuhlushwa kwe-enzyme: 1mg/mL

Fig. 5 Isitoreji uzinzilety at 25℃