Proteinase K NGS (pauta)
Numera pusi: HC4507A
NGS Protease K o se protease serine mautu ma gaoioiga enzyme maualuga ma lautele substrate specificity.The enzyme preferentially decomposes ester bonds ma peptide bonds e lata ane i le C-terminal of hydrophobic amino acids, sulfur-containing amino acids and aromatic amino acids.O lea la, e masani ona faʻaaogaina e faʻaleagaina ai polotini i peptides pupuu.NGS Protease K o se protease serine masani ma le Asp39-O lana69-Ser224catalytic triad lea e tulaga ese i serine proteases, ma o le nofoaga autu catalytic o loo siomia e tow Ca.2+nofoaga faʻapipiʻi mo le faʻamautuina, faʻatumauina le maualuga o le enzyme gaioiga i lalo o le lautele lautele o tulaga.
Fa'amatalaga
| Fa'aaliga | Pa'epa'e i le pa'epa'e pa'epa'e amorphous pauta, lyophilized |
| Gaoioiga faapitoa | ≥40U/mg malo |
| DNase | Leai se mea na iloa |
| RNase | Leai se mea na iloa |
| Avega olaola | ≤50CFU/g malo |
| Nucleic acid toega | <5pg/mg malo |
Meatotino
| Punavai | Tritirachium album |
| Numera EC | 3.4.21.64(Recombinant mai le Tritirachium album) |
| mamafa mole | 29kDa (SDS-PAGE) |
| Isoelectric point | 7.81 Ata.1 |
| pH sili ona lelei | 7.0-12.0 (O tagata uma e faʻatino gaioiga maualuga) Ata.2 |
| O le vevela sili ona lelei | 65℃ Ata.3 |
| pH mautu | pH 4.5-12.5 (25℃,16h) Ata.4 |
| Tulaga mafanafana | I lalo ole 50℃ (pH 8.0, 30min) Ata.5 |
| Tulaga mautu | Teu i le 25 ℃ mo le 12 masina Ata 6 |
| Activators | SDS, urea |
| Fa'alavelave | Diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Tulaga Teuteu
Teu le paʻu lyophilized i -25 ~ -15 ℃ mo se taimi umi mamao ese mai le malamalama;A maeʻa le faʻamavaeina, aliquot i totonu o le voluma talafeagai mo le teuina mo taimi pupuu i le 2-8 ℃ ese mai le malamalama poʻo le teuina umi i le -25 ~ -15 ℃ ese mai le malamalama.
Fa'aeteetega
Fai totigilima ma mata tioata pe a fa'aaoga pe mamafa, ma fa'amama lelei pe a uma ona fa'aoga.O lenei oloa e ono fa'atupu ai le fa'afefe o le pa'u ma fa'aleagaina mata.Afai e manava, e mafai ona mafua ai le allergy po'o fa'ailoga sela po'o le manava.E ono mafua ai le manava manava.
Fa'amatalaga o le iunite
E tasi le iunite ole NGS Protease K o loʻo faʻamatalaina o le aofaʻi o le enzyme e manaʻomia e faʻafefe ai le casein i le 1 μmol L-tyrosine i lalo o tulaga faʻavae masani.
Sauniuniga reagents
| Reagent | Tufuga | Fa'amaumauga |
| Casein tekinolosimai susu povi | Sigma Aldrich | C7078 |
| NaOH | Sinopharm ChemicalReagent Co., Ltd. | 10019762 |
| NaH2PO4·2H2O | Sinopharm ChemicalReagent Co., Ltd. | 20040718 |
| Na2HPO4 | Sinopharm ChemicalReagent Co., Ltd. | 20040618 |
| Trichloroacetic acid | Sinopharm ChemicalReagent Co., Ltd. | 80132618 |
| Sodium acetate | Sinopharm ChemicalReagent Co., Ltd. | 10018818 |
| Acetic acid | Sinopharm ChemicalReagent Co., Ltd. | 10000218 |
| HCl | Sinopharm ChemicalReagent Co., Ltd. | 10011018 |
| Sodium carbonate | Sinopharm ChemicalReagent Co., Ltd. | 10019260 |
| Foline-phenol | Sangon Biotech (Shanghai)Co., Ltd. | A500467-0100 |
| L-tyrosine | Sigma | 93829 |
Reagent I:
Faʻavae: 1% Casein mai le vaifofo susu povi: faʻamavae le 1g susu susu casein i le 50ml o le 0.1M sodium phosphate solution, pH 8.0, vevela ile vai taele ile 65-70 °C mo le 15mins, faʻafefe ma faʻamavae, faʻamafanafana i le vai, fetuunai e sodium hydroxide i le pH 8.0, ma faʻafefiloi i le 100ml.
Reagent II:
TCA vaifofo: 0.1M trichloroacetic acid, 0.2M sodium acetate ma 0.3M acetic acid (fua 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid sosoo, faaopoopo 50mL deionized vai, fetuutuunai ma le HCl3 i totonu o le HCl3. 100ml).
Reagent III:
0.4m le sodium carbonate solution (fuaina le 4.24g anhydrous sodium carbonate ma solo i le 100mL vai)
Reagent IV:
Folin phenol reagent: fa'alili fa'a 5 taimi ile vai fa'a'ona.
Reagent V:
Enzyme diluent: 0.1 M sodium phosphate solution, pH 8.0.
Reagent VI:
L-tyrosine fofo masani: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml L-tyrosine solo ma 0.2M HCl.
Taualumaga
1. Ia ki le UV-Vis spectrophotometer ma filifili le fua o le photometric.
2. Seti le galu e pei ole 660nm.
3. Liliu le vai taele, seti le vevela i le 37 ℃, faʻamautinoa e le suia le vevela mo 3-5mins.
4. Fa'avevela le mea'ai 0.5mL i totonu o se fa'amau fa'amaufa'ailoga 2mL i le vai taele 37℃ mo le 10mins.
5. Aveese le 0.5mL fa'afefeteina vaifofo fa'afefete i totonu o le fa'agaau centrifuge mua'i fa'avevela mo le 10 minute.Seti le enzyme diluent e fai ma vaega avanoa.
6. Fa'aopoopo le 1.0 mL TCA reagent i le taimi lava e uma ai le tali.Fa'afefiloi lelei ma fa'a'ofu i le ta'ele vai mo le 30 minute.
7. Centrifugate fofo tali.
8. Faʻaopoopo vaega nei i le faʻatonuga ua faʻamaonia.
| Reagent | Volume |
| Supernatant | 0.5 mL |
| 0.4M Sodium carbonate | 2.5 mL |
| Folin phenol reagent | 0.5 mL |
9. Fa'afefiloi lelei a'o le'i fa'a'ofuina ile ta'ele vai ile 37℃ mo le 30 min.
10. OD660na fuafuaina o le OD1;kulupu fa'atonu avanoa: E fa'aogaina le fa'amamafa o le enzyme e sui ai le vaifofo o le enzyme e fuafua ai le OD660pei ole OD2, ΔOD=OD1-OD2.
11. L-tyrosine curve masani: 0.5mL eseese concentration L-tyrosine fofo, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent i 5mL centrifuge tube, incubate i 37 ℃ mo 30mins, iloa mo OD660mo le fa'aogaina eseese o le L-tyrosine, ona maua lea o le fua fa'atatau Y=kX+b, o le Y o le L-tyrosine concentration, X o le OD.600.
Fa'atatauga
2: Aofa'i voluma o fofo tali (mL)
0.5: Volume ole vaifofo ole enzyme (mL)
0.5: Fa'aaogā le voluma vai fa'aaogā ile su'esu'ega chromogenic (mL)
10: Taimi e tali atu ai (min)
Df: Dilution tele
C: Fa'asa'o ole Enzyme (mg/mL)
Fa'atusa
Ata.1 DNA toega
| Fa'ata'ita'iga | Ave C4 | Nucleic Acid Toe fa'aleleia(pg/mg) | Toe fa'aleleia(%) | Aofa'i 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-Free H2O | Le mautinoa | — | — | — |
Ata.2 pH sili ona lelei
Ata.3 O le vevela sili ona lelei
Ata.4 pH Mausali
Ata.5 Mausalī vevela
Ata.6 Mausalī teuina ile 25℃
