Proteinase K mNGS (mmiri)
Proteinase K bụ serine protease kwụsiri ike nwere nkọwapụta mkpụrụ osisi sara mbara.Ọ na-eweda ọtụtụ protein na steeti ala ọbụna na ọnụnọ nke ncha.Ihe akaebe sitere na ọmụmụ ihe nhazi nke kristal na molecular na-egosi na enzyme bụ nke ezinụlọ subtilisin nwere triad catalytic saịtị na-arụ ọrụ (Asp).39-Ya69-Ser224).Ebe kachasị ebe mgbawa bụ njikọ peptide n'akụkụ otu carboxyl nke aliphatic na amino acid aromatic nwere otu alfa amino egbochiri.A na-ejikarị ya eme ihe maka nkọwa ya sara mbara.Emebere proteinase K nke a maka mNGS.E jiri ya tụnyere proteinase K nke ọzọ, ọ nwere ọbụna obere mmetọ nucleic acid nwere otu arụmọrụ enzymatic, nke nwere ike hụ na ngwa mNGS dị ala.
Ọnọdụ Nchekwa
2-8 ℃ maka afọ 2
Nkọwapụta
| Ọdịdị | Mmiri mmiri na-acha aja aja enweghị agba |
| Ihe omume | ≥800 U/ml |
| Ntinye uche protein | ≥20 mg/ml |
| Nickase | Ọnweghị nke achọpụtara |
| DNA | Ọnweghị nke achọpụtara |
| RNase | Ọnweghị nke achọpụtara |
Njirimara
| Nọmba EC | 3.4.21.64(Mgbakọ sitere na Tritirachium album) |
| Isi ihe nke isoelectric | 7.81 |
| pH kacha mma | 7.0-12.0 Foto 1 |
| Okpomọkụ kacha mma | 65 ℃ Fig. 2 |
| pH kwụsie ike | pH 4.5-12.5 (25 ℃, 16 h) Fig. 3 |
| Nkwụsi ike okpomọkụ | N'okpuru 50 ℃ (pH 8.0, 30 min) Fig. 4 |
| Nkwụsi ike nchekwa | Ihe karịrị 90% ọrụ maka ọnwa 12 na 25 ℃ |
| Ndị na-eme ihe | SDS, urea |
| Ihe mgbochi | Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Ngwa
1. Ngwa nyocha mkpụrụ ndụ ihe nketa
2. Ngwa RNA na DNA mmịpụta
3. Mwepu nke ihe ndị na-abụghị protein sitere na anụ ahụ, mmebi nke adịghị ọcha protein, dị ka DNA.ọgwụ mgbochi na nkwadebe nke heparin
4. Nkwadebe nke chromosome DNA site pulsed electrophoresis
5. Western blot
6. Enzymatic glycosylated albumin reagents in vitro nchoputa
Mkpachapụ anya
Yiri gloves nchebe na enyo mgbe ị na-eji ma ọ bụ na-atụ aro, ma na-ekuku ikuku nke ọma mgbe ejiri ya.Ngwaahịa a nwere ike ibute mmeghachi ahụ nfụkasị anụ ahụ yana oke iwe anya.Ọ bụrụ na ikuru ume, ọ nwere ike ibute nfụkasị ahụ ma ọ bụ mgbaàmà ụkwara ume ọkụ ma ọ bụ dyspnea.Nwere ike ịkpata mgbakasị iku ume.
Nkọwa nkeji
A kọwapụtara otu nkeji (U) dị ka ọnụọgụ enzyme achọrọ iji hydrolyze casein iji mepụta 1 μmol.tyrosine kwa nkeji n'okpuru ọnọdụ ndị a.
Reagents nkwadebe
Reagent I: 1g mmiri ara ehi casein gbazere na 50ml nke 0.1M sodium phosphate solution (pH 8.0), etinyere ya na 65-70 ℃ mmiri maka 15mins, kpalitere ma gbazee, mee ka mmiri dị jụụ, nke sodium hydroxide gbanwere na pH 8.0, na olu edoziri. 100ml.
Reagent II: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
Reagent III: 0.4M Na2CO3ngwọta.
Reagent IV: Forint reagent ejiri mmiri dị ọcha gbazere ugboro ise.
Reagent V: Enzyme diluent: 0.1M sodium phosphate ngwọta (pH 8.0).
Reagent VI: ngwọta tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine etisasịwo na 0.2M HCl.
Usoro
1. 0.5ml nke reagent m bụ pre-warmed ka 37 ℃, tinye 0.5ml nke enzyme ngwọta, mix ọma, na incubate na37 ℃ maka nkeji 10.
2. Tinye 1ml nke reagent II iji kwụsị mmeghachi omume ahụ, gwakọta nke ọma, ma nọgide na-emepụta ihe maka 30mins.
3. Centrifugate mmeghachi omume ngwọta.
4. Were 0.5ml supernatant, tinye 2.5ml reagent III, 0.5ml reagent IV, mix ọma na incubate na 37 ℃maka 30mins.
5. OD660e kpebisiri ike dị ka OD1;Otu njikwa oghere: 0.5ml reagent V na-eji dochie enzymengwọta iji chọpụta OD660dị ka OD2, ΔOD=OD1-Od2.
6. L-tyrosine ọkọlọtọ usoro: 0.5mL iche iche ịta L-tyrosine ngwọta, 2.5mL Reagent III, 0.5mL Reagent IV na 5mL centrifuge tube, incubate na 37 ℃ maka 30mins, chọpụta maka OD660maka ọkwa dị iche iche nke L-tyrosine, wee nweta usoro ọkọlọtọ Y = kX + b, ebe Y bụ ntinye L-tyrosine, X bụ OD.600.
Mgbakọ
2: ngụkọta olu ngwọta mmeghachi omume (mL)
0.5: Olu nke ngwọta enzyme (mL)
0.5: Mmeghachi omume olu mmiri mmiri ejiri na mkpebi chromogenic (mL)
10: Oge mmeghachi omume (nkeji)
Df: Dilution otutu
CNtinye nke enzymes (mg/mL)
Ntụaka
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77 .
2. Wieger U & Hilz H. Biochem.Biophys.Res.Kọmun.(1971);44:513 .
3. Hilz, H.et al.,Euro.J. Biochem.(1975);56:103–108 .
4. Sambrook Jet al., Cloning Molecular: Akwụkwọ ntuziaka ụlọ nyocha, mbipụta nke abụọ, ọdụ ụgbọ mmiri oyiLaboratory Press, Cold Spring Harbor (1989).
Ọnụọgụ
fig.1 Kachasị mma pH
Ngwọta nchekwa 100mM: pH6.0-8.0, Na-phosphate;pH8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Enzyme itinye uche: 1mg/mL
Fig.2 okpomọkụ kacha mma
Mmeghachi omume na 20 mM K-phosphate buffer pH 8.0.Ntinye uche nke enzyme: 1 mg/mL
Fig.3 pH Nkwụsi ike
25 ℃, 16 h-ọgwụgwọ na 50 mm buffer ngwọta: 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.Ntinye uche nke enzyme: 1 mg/mL
Fig.4 Thermal nkwụsi ike
Ọgwụgwọ 30 min nwere ihe nchekwa 50mM Tris-HCl, pH 8.0.Ntinye uche nke enzyme: 1 mg/mL
Fig.5 Nchekwa kwụsie ikety at 25 ℃
