Proteinase K mNGS (omi)
Proteinase K jẹ protease serine iduroṣinṣin pẹlu pato sobusitireti gbooro.O degrades ọpọlọpọ awọn ọlọjẹ ni ilu abinibi ipinle ani niwaju detergents.Ẹri lati awọn iwadii igbekalẹ kirisita ati ti molikula tọka pe enzymu jẹ ti idile subtilisin pẹlu triad catalytic aaye ti nṣiṣe lọwọ (Asp39-Tirẹ69-Ser224).Aaye pataki ti cleavage ni asopọ peptide ti o wa nitosi ẹgbẹ carboxyl ti aliphatic ati awọn amino acid ti oorun didun pẹlu awọn ẹgbẹ alpha amino ti dina.O ti wa ni commonly lo fun awọn oniwe-gbooro pato.proteinase K yii jẹ apẹrẹ pataki fun mNGS.Ti a ṣe afiwe pẹlu proteinase K miiran, o ni paapaa ibajẹ acid nucleic ti o kere si pẹlu iṣẹ enzymatic kanna, eyiti o le rii daju pe ohun elo mNGS isalẹ isalẹ.
Awọn ipo ipamọ
2-8 ℃ fun ọdun meji
Sipesifikesonu
| Ifarahan | Aila-awọ si omi alawọ brown |
| Iṣẹ-ṣiṣe | ≥800 U/ml |
| Ifojusi Amuaradagba | ≥20 mg/ml |
| Nickase | Ko si ọkan ti a rii |
| DNA | Ko si ọkan ti a rii |
| RNase | Ko si ọkan ti a rii |
Awọn ohun-ini
| EC nọmba | 3.4.21.64(Atunṣe lati awo-orin Tritirachium) |
| Isoelectric ojuami | 7.81 |
| pH ti o dara julọ | 7.0-12.0 olusin 1 |
| Iwọn otutu to dara julọ | 65 ℃ aworan 2 |
| pH iduroṣinṣin | pH 4.5-12.5 (25 ℃, 16 h) eeya 3 |
| Iduroṣinṣin gbona | Ni isalẹ 50 ℃ (pH 8.0, 30 min) eeya. 4 |
| Iduroṣinṣin ipamọ | Ju 90% iṣẹ ṣiṣe fun oṣu 12 ni 25 ℃ |
| Awọn aṣiṣẹ | SDS, urea |
| Awọn oludena | Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Awọn ohun elo
1. Jiini okunfa kit
2. RNA ati DNA isediwon irin ise
3. Iyọkuro awọn ohun elo ti kii ṣe amuaradagba lati awọn tisọ, ibajẹ ti awọn idoti amuaradagba, gẹgẹbi DNAAjẹsara ati igbaradi ti heparin
4. Igbaradi ti chromosome DNA nipasẹ pulsed electrophoresis
5. Western abawọn
6. Enzymatic glycosylated albumin reagents in vitro okunfa
Àwọn ìṣọ́ra
Wọ awọn ibọwọ aabo ati awọn goggles nigba lilo tabi ṣe iwọn, ki o jẹ afẹfẹ daradara lẹhin lilo.Ọja yii le fa ifa inira awọ ara ati híhún oju to ṣe pataki.Ti a ba fa simu, o le fa aleji tabi awọn ami ikọ-fèé tabi dyspnea.Le fa ibinu ti atẹgun.
Itumọ ẹyọkan
Ẹyọ kan (U) jẹ asọye bi iye henensiamu ti o nilo lati hydrolyze casein lati ṣe agbejade 1 μmoltyrosine fun iṣẹju kan labẹ awọn ipo wọnyi.
Reagents igbaradi
Reagent I: 1g wara casein ti tuka ni 50ml ti 0.1M sodium phosphate solution (pH 8.0), ti a fi sinu omi 65-70 ℃ fun awọn iṣẹju 15, rú ati tituka, tutu nipasẹ omi, ṣatunṣe nipasẹ sodium hydroxide si pH 8.0, ati iwọn didun ti o wa titi 100ml.
Reagent II: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
Reagent III: 0.4M Na2CO3ojutu.
Reagent IV: Forint reagent ti fomi po pẹlu omi mimọ fun awọn akoko 5.
Reagent V: Diluent Enzyme: 0.1M iṣuu soda fosifeti ojutu (pH 8.0).
Reagent VI: ojutu tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine tituka pẹlu 0.2M HCl.
Ilana
1. 0.5ml ti reagent I ti wa ni iṣaju si 37℃, fi 0.5ml ti ojutu enzymu kun, dapọ daradara, ki o si ṣafikun ni37 ℃ fun iṣẹju 10.
2. Fi 1ml ti reagent II lati da iṣesi duro, dapọ daradara, ki o tẹsiwaju abeabo fun 30mins.
3. Centrifugate lenu ojutu.
4. Mu 0.5ml supernatant, fi 2.5ml reagent III, 0.5ml reagent IV, dapọ daradara ati incubate ni 37℃fun 30 iṣẹju.
5. OD660ti pinnu bi OD1;òfo Iṣakoso ẹgbẹ: 0.5ml reagent V ti lo lati ropo henensiamuojutu lati pinnu OD660bi OD2, ΔOD=OD1-OD2.
6. L-tyrosine boṣewa ti tẹ: 0.5mL iyatọ ifọkansi L-tyrosine ojutu, 2.5mL Reagent III, 0.5mL Reagent IV ni tube centrifuge 5mL, incubate ni 37 ℃ fun 30mins, ṣawari fun OD660fun oriṣiriṣi ifọkansi ti L-tyrosine, lẹhinna gba iwọn boṣewa Y = kX + b, nibiti Y jẹ ifọkansi L-tyrosine, X jẹ OD600.
Iṣiro
2: Lapapọ iwọn didun ti ojutu esi (ml)
0.5: Iwọn ti ojutu enzymu (mL)
0.5: Iwọn omi ifasẹyin ti a lo ninu ipinnu chromogenic (mL)
10: Àkókò ìdáhùn (iṣẹ́jú)
Df: Dilution ọpọ
CIfojusi Enzyme (mg/ml)
Awọn itọkasi
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.et al.,Euro.J. Biokemu.(1975);56:103–108.
4. Sambrook Jet al., Cloning Molecular: Afọwọṣe yàrá kan, ẹda 2nd, Harbor Orisun omi tutuYàrá Tẹ, Tutu Orisun omi Harbor (1989).
Awọn isiro
eeya.1 O dara julọ pH
100mM ojutu ifipamọ: pH6.0-8.0, Na-fosifeti;pH8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Enzyme fojusi: 1mg/ml
Fig.2 Iwọn otutu to dara julọ
Idahun ni 20 mM K-fosifeti ifipamọ pH 8.0.Ifojusi Enzymu: 1 mg/ml
aworan.3 pH Iduroṣinṣin
25 ℃, 16 h-itọju pẹlu 50 mM ojutu ifipamọ: pH 4.5- 5.5, Acetate;pH 6.0-8.0, Na-fosifeti;pH 8.0-9.0, Tris-HCl.pH 9.0- 12.5, Glycine-NaOH.Ifojusi Enzymu: 1 mg/ml
Fig.4 Gbona iduroṣinṣin
30 min-itọju pẹlu 50 mM Tris-HCl ifipamọ, pH 8.0.Ifojusi Enzymu: 1 mg/ml
Fig.5 Ibi ipamọ iduroṣinṣinty at 25 ℃
