Proteinase K (Omi)
Nọmba ologbo: HC4502A
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-gbooropato.
Sipesifikesonu
| Ifarahan | Aila-awọ si omi alawọ brown |
| Iṣẹ-ṣiṣe | ≥800 U/ml |
| Amuaradagba ifọkansi | ≥20 mg/ml |
| DNA | Ko si ọkan ti a rii |
| RNase | Ko si ọkan ti a rii |
Awọn ipo ipamọ
Fipamọ ni iwọn otutu ti 2-8 ℃.
Awọn ohun-ini
| EC nọmba | 3.4.21.64 (Recombinant lati Tritirachium album) |
| Ìwúwo molikula | 29 kDa (SDS-iwe) |
| Isoelectric ojuami | 7.81 |
| pH ti o dara julọ | 7.0-12.0 olusin.1 |
| Iwọn otutu to dara julọ | 65 ℃ Fig.2 |
| pH iduroṣinṣin | pH 4.5-12.5 (25 ℃, 16 h) Fig.3 |
| Iduroṣinṣin gbona | Ni isalẹ 50 ℃ (pH 8.0, 30 mins) Fig.4 |
| 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 ti awọn ohun elo ti kii ṣe amuaradagba lati awọn tisọ, ibajẹ ti awọn ohun elo amuaradagba, gẹgẹbi awọn ajesara DNA 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.
Ayẹwo
Itumọ ẹyọkan
Ẹyọ kan (U) jẹ asọye bi iye henensiamu ti o nilo lati ṣe hydrolyze casein lati ṣe agbejade 1 μmol tyrosine fun iṣẹju kan labẹ awọn ipo atẹle.
Reagents igbaradi
Reagent I: 1g wara casein ti ni tituka 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 pH8.0, ati ti o wa titi iwọn didun 100ml.
Reagent II: ojutu TCA: 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, ati incubate ni 37 ℃ fun 10mins.
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 30mins.
5. OD660ti pinnu bi OD1;ẹgbẹ iṣakoso ofo: 0.5ml reagent V ti lo lati rọpo ojutu enzymu 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, àtúnse 2nd, Cold Spring Harbor Laboratory Press, Cold Spring 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
olusin 2 Ti o dara ju temperatur
Idahun ni 20mM K-fosifeti ifipamọ pH 8.0.Ifojusi Enzymu: 1mg/ml
olusin 3 pH Iduroṣinṣin
25℃,16 h-itọju pẹlu 50mM 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: 1mg/ml
olusin 4 Gbona iduroṣinṣin
30 min-itọju pẹlu 50mM Tris-HCl ifipamọ, pH 8.0.Ifojusi Enzymu: 1mg/ml
olusin 5 Ibi ipamọ iduroṣinṣinty at 25 ℃
