Proteinase K NGS (lulú)
Nọmba ologbo: HC4507A
NGS Protease K ni a idurosinsin serine protease pẹlu ga henensiamu aṣayan iṣẹ-ṣiṣe ati jakejado sobusitireti Specificity.The henensiamu preferentially decomposes ester iwe adehun ati peptide ìde nitosi si C-terminal ti hydrophobic amino acids, efin-ti o ni awọn amino acids ati oorun didun amino acids.Nitorinaa, igbagbogbo lo lati dinku awọn ọlọjẹ sinu awọn peptides kukuru.NGS Protease K jẹ aṣoju serine protease pẹlu Asp39-Tirẹ69-Ser224triad catalytic eyiti o jẹ alailẹgbẹ si awọn proteases serine, ati ile-iṣẹ katalitiki ti yika nipasẹ gbigbe Ca.2+awọn aaye abuda fun imuduro, mimu iṣẹ ṣiṣe enzymu giga labẹ ọpọlọpọ awọn ipo.
Sipesifikesonu
| Ifarahan | Funfun si pa-funfun amorphous lulú, lyophilized |
| Iṣẹ ṣiṣe pato | ≥40U/mg ti o lagbara |
| DNA | Ko si ọkan ti a rii |
| RNase | Ko si ọkan ti a rii |
| Ẹru-ẹmi | ≤50CFU/g ti o lagbara |
| Ajẹkù acid Nucleic | <5pg/mg ti o lagbara |
Awọn ohun-ini
| Orisun | Tritirachium awo-orin |
| EC nọmba | 3.4.21.64(Atunṣe lati awo-orin Tritirachium) |
| Ìwúwo molikula | 29kDa (SDS-iwe) |
| Isoelectric ojuami | 7,81 olusin.1 |
| pH ti o dara julọ | 7.0-12.0 (Gbogbo ṣe ga akitiyan) Fig.2 |
| Iwọn otutu to dara julọ | 65℃ Fig.3 |
| pH Iduroṣinṣin | pH 4.5-12.5 (25 ℃, 16h) Fig.4 |
| Iduroṣinṣin gbona | Ni isalẹ 50 ℃ (pH 8.0, 30min) Fig.5 |
| Iduroṣinṣin ipamọ | Ti a fipamọ ni 25℃ fun awọn oṣu 12 Fig.6 |
| Awọn aṣiṣẹ | SDS, urea |
| Awọn oludena | Diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Awọn ipo ipamọ
Tọju lulú lyophilized ni -25 ~ -15 ℃ fun igba pipẹ kuro lati ina;Lẹhin itusilẹ, aliquot sinu iwọn ti o yẹ fun ibi ipamọ igba kukuru ni 2-8℃ kuro lati ina tabi ibi ipamọ igba pipẹ ni -25 ~ -15 ℃ kuro lati ina.
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 ti NGS Protease K jẹ asọye bi iye henensiamu ti o nilo lati ṣe hydrolyze casein sinu 1 μmol L-tyrosine labẹ awọn ipo ipinnu boṣewa.
Reagents igbaradi
| Reagent | Olupese | Katalogi |
| Casein imọlati wara ẹran | Sigma Aldrich | C7078 |
| NÁOH | Sinopharm KemikaliReagent Co., Ltd. | Ọdun 10019762 |
| NAH2PO4· 2H2O | Sinopharm KemikaliReagent Co., Ltd. | Ọdun 20040718 |
| N2HPO4 | Sinopharm KemikaliReagent Co., Ltd. | Ọdun 20040618 |
| Trichloroacetic acid | Sinopharm KemikaliReagent Co., Ltd. | 80132618 |
| Iṣuu soda acetate | Sinopharm KemikaliReagent Co., Ltd. | Ọdun 10018818 |
| Acid acid | Sinopharm KemikaliReagent Co., Ltd. | 10000218 |
| HCl | Sinopharm KemikaliReagent Co., Ltd. | 10011018 |
| Sodium kaboneti | Sinopharm KemikaliReagent Co., Ltd. | Ọdun 10019260 |
| Foline-phenol | Sangon Biotech (Shanghai)Co., Ltd. | A500467-0100 |
| L-tyrosini | Sigma | 93829 |
Reagent I:
Sobusitireti: 1% Casein lati ojutu wara bovine: tu 1g wara bovine casein ni 50ml ti 0.1M sodium phosphate solution, pH 8.0, ooru ninu iwẹ omi ni 65-70 °C fun 15mins, aruwo ati tu, dara pẹlu omi, ti a ṣatunṣe nipasẹ iṣuu soda hydroxide si pH 8.0, ati dilute sinu si 100ml.
Reagent II:
Ojutu TCA: 0.1M trichloroacetic acid, 0.2M sodium acetate ati 0.3M acetic acid (ṣe iwọn 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid leralera, ṣafikun 50mL deionized omi, ṣatunṣe pẹlu HCl si pH 4.3H. 100 milimita).
Reagent III:
0.4m iṣuu soda kaboneti ojutu (ṣe iwọn 4.24g iṣuu soda carbonate anhydrous ati tu ni omi 100mL)
Reagent IV:
Folin phenol reagent: dilute awọn akoko 5 pẹlu omi deionized.
Reagent V:
Diluent enzymu: 0.1 M iṣuu soda fosifeti ojutu, pH 8.0.
Reagent VI:
Ojutu boṣewa L-tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml L-tyrosine tituka pẹlu 0.2M HCl.
Ilana
1. Tan UV-Vis spectrophotometer ki o yan wiwọn photometric.
2. Ṣeto awọn wefulenti bi 660nm.
3. Tan-an omi iwẹ, ṣeto iwọn otutu si 37 ℃, rii daju pe iwọn otutu ko yipada fun 3-5mins.
4. Ṣaju sobusitireti 0.5mL ni tube centrifuge 2mL ni 37℃ omi iwẹ fun 10mins.
5. Jade 0.5mL ti fomi ojutu enzymu sinu tube centrifuge ti a ti ṣaju fun awọn iṣẹju 10.Ṣeto diluent henensiamu bi ẹgbẹ òfo.
6. Fi 1.0 milimita TCA reagent kun lẹsẹkẹsẹ lẹhin iṣesi.Illa daradara ati incubate ninu omi wẹ fun ọgbọn išẹju 30.
7. Centrifugate lenu ojutu.
8. Fi awọn wọnyi irinše ni awọn ibere pàtó kan.
| Reagent | Iwọn didun |
| Alabojuto | 0,5 milimita |
| 0.4M iṣuu soda kaboneti | 2.5 milimita |
| Folin phenol reagent | 0,5 milimita |
9. Illa daradara ṣaaju ki o to incubating ni omi wẹ a 37 ℃ fun 30 mins.
10. OD660ti pinnu bi OD1;Ẹgbẹ iṣakoso òfo: Diluent Enzyme ti lo lati rọpo ojutu enzymu lati pinnu OD660bi OD2, ΔOD=OD1-OD2.
11. L-tyrosine boṣewa ti tẹ: 0.5mL o yatọ si ifọkansi L-tyrosine ojutu, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent ni 5mL centrifuge tube, incubate ni 37 ℃ fun 30mins, ri fun OD660fun oriṣiriṣi ifọkansi ti L-tyrosine, lẹhinna gba ọna kika 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 isiro
Fig.1 DNA aloku
| Apeere | Ave C4 | Nucleic Acid Imularada(pg/mg) | Imularada(%) | Lapapọ Nucleic Acid ( pg/mg) |
| PRK | 24.66 | 2.23 | 83% | 2.687 |
| PRK+STD2 | 18.723 | 126.728 | - | - |
| STD1 | 12.955 |
- |
- |
- |
| STD2 | 16 | |||
| STD3 | 19.125 | |||
| STD4 | 23.135 | |||
| STD5 | 26.625 | |||
| RNA-Ọfẹ H2O | Ti a ko pinnu | - | - | - |
Fig.2 Ti o dara ju pH
Fig.3 Iwọn otutu to dara julọ
Fig.4 pH Iduroṣinṣin
Fig.5 Gbona iduroṣinṣin
Fig.6 Iduroṣinṣin ipamọ ni 25 ℃
