Proteinase K NGS (budada)
Bisadda No: HC4507A
NGS Protease K waa serine protease xasiloon oo leh hawl enzyme sare iyo Specific substrate ballaaran.Ensaym doorbidayaa decomposes ester bonds iyo bonds peptide ku dheggan C-terminal ee hydrophobic amino acids, sulfur-ka kooban amino acids iyo amino acids caraf udgoon.Sidaa darteed, waxaa badanaa loo isticmaalaa in lagu yareeyo borotiinada peptides gaaban.NGS Protease K waa protease serine caadi ah oo leh Asp39-Kiisa69- Ser224Katalytic triad kaas oo u gaar ah serine proteases, iyo xarunta catalytic waxaa ku hareeraysan jiid Ca2+goobaha lagu xirayo xasilinta, ilaalinta dhaqdhaqaaqa enzyme sare ee hoos yimaada xaalado kala duwan.
Tilmaamid
| Muuqashada | Caddaan ama budo amorphous ah, lyophilized |
| Dhaqdhaqaaq gaar ah | ≥40U/mg adag |
| DNA | Midna lama ogaan |
| RNase | Midna lama ogaan |
| Culayska noole | ≤50CFU/g adag |
| Haraaga aashitada nucleic | <5pg/mg adag |
Guryaha
| Xigasho | Tritirachium |
| lambarka EC | 3.4.21.64(Recombinant from Tritirachium album) |
| Miisaanka molecular | 29kDa (SDS-BOGGA) |
| Barta koronto | 7.81 Jaantus.1 |
| pH ugu fiican | 7.0-12.0 (Dhammaan waxay qabtaan hawlo sare) Jaantus.2 |
| Heerkulka ugu wanaagsan | 65℃ Jaantus.3 |
| Xasiloonida pH | pH 4.5-12.5 (25℃,16h) Jaantus.4 |
| Deganaanshaha kulaylka | Ka hooseeya 50 ℃ (pH 8.0, 30min) Jaantus.5 |
| xasiloonida kaydinta | Waxa lagu kaydiyaa 25℃ 12 bilood Jaantuska.6 |
| Dhaqdhaqaaqayaasha | SDS, urea |
| Kahortagayaasha | Diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Xaaladaha Kaydinta
Ku kaydi budada lyophilized at -25 ~ -15 ℃ wakhti dheer ka fog iftiinka;Kala diri ka dib, u gudbi mugga ku habboon kaydinta muddada-gaaban ee 2-8℃ ka fog iftiinka ama kaydinta muddada-dheer ee -25 ~ -15 ℃ meel ka fog iftiinka.
Ka taxadaritaan
Xidho galoofyada ilaalinta iyo muraayadaha marka aad isticmaalayso ama miisaanayso, si fiicanna hawo u sii isticmaal ka dib.Alaabtani waxay keeni kartaa fal-celin xasaasiyad maqaarka ah iyo cuncun daran oo indhaha ah.Haddii la nuugo, waxay keeni kartaa xasaasiyad ama calaamadaha neefta ama dyspnea.Waxa laga yaabaa inay keento cuncun xagga neefsiga ah.
Qeexitaanka unugga
Hal cutub oo ka mid ah NGS Protease K waxaa lagu qeexaa sida qadarka ensaymka looga baahan yahay in lagu hydrolyze casein galay 1 μmol L-tyrosine hoos xaaladaha go'aaminta caadiga ah.
Reagents diyaarinta
| Reagent | Soo saaraha | Buug-gacmeedka |
| Casein farsamocaanaha lo'da | Sigma Aldrich | C7078 |
| NaOH | Kiimikada SinopharmHal qayb oo ka mid ah Reagent Co., Ltd. | 10019762 |
| NaH2PO4·2H2O | Kiimikada SinopharmHal qayb oo ka mid ah Reagent Co., Ltd. | 20040718 |
| Na2HPO4 | Kiimikada SinopharmHal qayb oo ka mid ah Reagent Co., Ltd. | 20040618 |
| Trichloroacetic acid | Kiimikada SinopharmHal qayb oo ka mid ah Reagent Co., Ltd. | 80132618 |
| Sodium acetate | Kiimikada SinopharmHal qayb oo ka mid ah Reagent Co., Ltd. | 10018818 |
| Acid acid | Kiimikada SinopharmHal qayb oo ka mid ah Reagent Co., Ltd. | 10000218 |
| HCl | Kiimikada SinopharmHal qayb oo ka mid ah Reagent Co., Ltd. | 10011018 |
| Sodium carbonate | Kiimikada SinopharmHal qayb oo ka mid ah Reagent Co., Ltd. | 10019260 |
| Foline-phenol | Sangon Biotech (Shanghai)Co., Ltd. | A500467-0100 |
| L-tyrosine | Sigma | 93829 |
Reagent I:
Substrate: 1% Casein laga bilaabo caanaha lo'da: milaan 1g caanaha lo'da casein ee 50ml of 0.1M sodium phosphate solution, pH 8.0, kulaylka biyaha qubeyska 65-70 °C 15mins, walaaq oo milaan, ku qabooji biyo, lagu hagaajiyay sodium hydroxide ilaa pH 8.0, ku milmi ilaa 100ml.
Reagent II:
Xalka TCA: 0.1M trichloroacetic acid, 0.2M sodium acetate iyo 0.3M acetic acid (miisaanka 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid si isdaba joog ah, ku dar 50mL biyaha deionized, ku hagaaji HCl ilaa 4H. 100ml).
Reagent III:
0.4m sodium carbonate xal (miisaanka 4.24g anhydrous sodium carbonate iyo milaan 100mL biyo)
Reagent IV:
Folin phenol reagent: ku qas 5 jeer biyo deionized ah.
Reagent V:
Isku-darka Enzyme: 0.1 M sodium phosphate solution, pH 8.0.
Reagent VI:
Xalka caadiga ah ee L-tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml L-tyrosine oo lagu milmay 0.2M HCl.
Habraaca
1. Daar UV-Vis spectrophotometer oo dooro cabbirka sawir-qaadista.
2. U deji hirarka dhererka 660nm.
3. Daar qubayska biyaha, dhig heerkulka 37℃, hubi in heerkulku aanu isbeddelin 3-5mins.
4. Ku sii kululee 0.5mL substrate ku jira tuubo centrifuge 2ml at 37℃ qubeyska biyaha 10mins.
5. Ka soo saar 0.5mL xal enzyme la badhxay tuubada centrifuge preheated ilaa 10 daqiiqo.U deji dareeraha enzyme sida koox madhan.
6. Ku dar 1.0 ml TCA reagent isla markiiba ka dib falcelinta.Si fiican isku qas oo ku rid biyaha qubeyska 30 daqiiqo.
7. Xalka falcelinta centrifugate.
8. Ku dar qaybaha soo socda sida ay u kala horreeyaan.
| Reagent | Mugga |
| Sareeye | 0.5 ml |
| 0.4M Sodium carbonate | 2.5 ml |
| Folin phenol reagent | 0.5 ml |
9. Si fiican isku qas ka hor inta aanad ku dhex gelin biyaha qubeyska 37 ℃ 30 daqiiqo.
10. OD660waxaa loo go'aamiyay sida OD1;Kooxda xakamaynta bannaan: Diluent Enzyme waxaa loo isticmaalaa in lagu beddelo xalka enzyme si loo go'aamiyo OD660sida OD2, ΔOD=OD1-OD2.
11. L-tyrosine qalooca caadiga ah: 0.5mL fiirsashada kala duwan L-tyrosine xal, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent in tube centrifuge 5mL, incubate in 37 ℃ 30mins, ogaado OD660fiirsashada kala duwan ee L-tyrosine, ka dibna la helay qalooca caadiga ah Y = kX + b, halkaasoo Y ay tahay xoojinta L-tyrosine, X waa OD600.
Xisaabinta
2: Wadarta guud ee xalalka falcelinta (ml)
0.5: Mugga xalinta enzyme (mL)
0.5: Mugga dareeraha falcelinta loo isticmaalo go'aaminta chromogenic (mL)
10: Waqtiga falcelinta (min)
Df: Kala-saar badan
CXakamaynta enzyme (mg/mL)
Sawirro
Jaantuska.1 hadhaaga DNA
| Tusaale | Ave C4 | Nucleic acid Soo kabashada (pg/mg) | Soo kabashada(%) | Wadarta Nukliyeerka Aashito ( 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-H2O bilaash ah | Lama go'aamin | - | - | - |
Jaantuska.2 pH ugu fican
Jaantus.3 Heerkulka ugu wanaagsan
Jaantuska.4 pH Xasiloonida
Jaantus.5 Deganaanshaha kulaylka
Jaantuska.6 Xasiloonida kaydinta at 25℃
