Proteinase K NGS (foda)
Saukewa: HC4507A
NGS Protease K ne barga serine protease tare da high enzyme aiki da kuma m substrate specificity.The enzyme preferentially decomposes ester bond da peptide bonds dab da C-tasha na hydrophobic amino acid, sulfur-dauke da amino acid da aromatic amino acid.Don haka, ana amfani da shi sau da yawa don lalata sunadarai zuwa gajerun peptides.NGS Protease K shine nau'in protease na yau da kullun tare da Asp39-Nasa69-Ser224triad catalytic wanda ya keɓanta ga serine proteases, kuma cibiyar catalytic tana kewaye da tow Ca.2+wuraren dauri don daidaitawa, kiyaye babban aikin enzyme a ƙarƙashin yanayi mai faɗi.
Ƙayyadaddun bayanai
| Bayyanar | Fari zuwa kashe-fari amorphous foda, lyophilized |
| takamaiman ayyuka | ≥40U/mg mai ƙarfi |
| DNA | Babu wanda aka gano |
| RNase | Babu wanda aka gano |
| Kwayoyin halitta | ≤50CFU/g m |
| Nucleic acid ragowar | <5pg/mg mai ƙarfi |
Kayayyaki
| Source | Tritirachium album |
| lambar EC | 3.4.21.64(Recombinant daga Tritirachium album) |
| Nauyin kwayoyin halitta | 29kDa (SDS-SHAFIN) |
| Isoelectric batu | 7.81 Hoto 1 |
| Mafi kyawun pH | 7.0-12.0 (Duk suna yin babban aiki) Hoto 2 |
| Mafi kyawun zafin jiki | 65℃ Hoto.3 |
| pH Stability | pH 4.5-12.5 (25 ℃, 16h) Hoto.4 |
| Zaman lafiyar thermal | Kasa 50 ℃ (pH 8.0, 30min) Hoto.5 |
| kwanciyar hankali na ajiya | Ajiye a 25 ℃ na watanni 12 Hoto.6 |
| Masu kunnawa | SDS, urea |
| Masu hanawa | Diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Yanayin Ajiya
Ajiye lyophilized foda a -25 ~ -15 ℃ na dogon lokaci daga haske;Bayan narkar da, aliquot cikin dace girma ga gajere ajiya a 2-8 ℃ daga haske ko dogon lokacin ajiya a -25 ~ -15 ℃ daga haske.
Matakan kariya
Saka safofin hannu masu kariya da tabarau lokacin amfani ko aunawa, kuma kiyaye iska sosai bayan amfani.Wannan samfurin na iya haifar da rashin lafiyar fata da kuma tsananin haushin ido.Idan an shaka, yana iya haifar da alerji ko alamun asma ko dyspnea.Zai iya haifar da haushin numfashi.
Ma'anar raka'a
Ɗayan raka'a na NGS Protease K an bayyana shi azaman adadin enzyme da ake buƙata don hydrolyze casein zuwa 1 μmol L-tyrosine a ƙarƙashin daidaitaccen yanayin ƙaddara.
Reagents shiri
| Reagent | Mai ƙira | Katalogi |
| Casein fasahadaga madarar nono | Sigma Aldrich | C7078 |
| NaOH | Sinopharm ChemicalReagent Co., Ltd. | 10019762 |
| NaH2PO4· 2H2O | Sinopharm ChemicalReagent Co., Ltd. | 20040718 |
| Farashin 2HPO4 | Sinopharm ChemicalReagent Co., Ltd. | 20040618 |
| Trichloroacetic acid | Sinopharm ChemicalReagent Co., Ltd. | 80132618 |
| Sodium acetate | Sinopharm ChemicalReagent Co., Ltd. | Farashin 10018818 |
| Acetic acid | Sinopharm ChemicalReagent Co., Ltd. | 10000218 |
| HCl | Sinopharm ChemicalReagent Co., Ltd. | Farashin 10011018 |
| Sodium carbonate | Sinopharm ChemicalReagent Co., Ltd. | Farashin 10019260 |
| Foline-phenol | Sangon Biotech (Shanghai)Co., Ltd. | Saukewa: A500467-0100 |
| L-tyrosine | Sigma | 93829 |
Reagent I:
Substrate: 1% Casein daga ruwan madara na bovine: narke 1g madarar nono casein a cikin 50ml na 0.1M sodium phosphate solution, pH 8.0, zafi a cikin wanka na ruwa a 65-70 ° C na 15mins, motsawa kuma narke, sanyi da ruwa, daidaitacce ta sodium hydroxide zuwa pH 8.0, kuma tsarma a cikin 100ml.
Reagent II:
Maganin TCA: 0.1M trichloroacetic acid, 0.2M sodium acetate da 0.3M acetic acid (auna 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid jere, ƙara 50mL deionized ruwa, daidaita tare da HCl zuwa p. 100 ml).
Reagent III:
0.4m sodium carbonate bayani (nauyin 4.24g anhydrous sodium carbonate da narke a cikin 100ml ruwa)
Reagent IV:
Folin phenol reagent: tsarma sau 5 tare da ruwa mai narkewa.
Reagent V:
Enzyme diluent: 0.1 M sodium phosphate bayani, pH 8.0.
Reagent VI:
L-tyrosine daidaitaccen bayani: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml L-tyrosine narkar da 0.2M HCl.
Tsari
1. Kunna UV-Vis spectrophotometer kuma zaɓi ma'aunin hoto.
2. Saita tsawon zangon kamar 660nm.
3. Kunna ruwan wanka, saita zafin jiki zuwa 37 ℃, tabbatar da zafin jiki ba canzawa don 3-5mins.
4. Preheat 0.5mL substrate a cikin wani 2mL centrifuge tube a 37 ℃ ruwa wanka for 10mins.
5. Cire 0.5mL diluted enzyme bayani a cikin preheated centrifuge bututu na minti 10.Saita diluent enzyme a matsayin rukunin mara komai.
6. Ƙara 1.0 ml TCA reagent nan da nan bayan amsawa.Mix sosai kuma a saka a cikin ruwan wanka na tsawon minti 30.
7. Centrifugate dauki bayani.
8. Ƙara abubuwan da ke biyo baya a cikin tsari da aka ƙayyade.
| Reagent | Ƙarar |
| Mai girma | 0.5 ml |
| 0.4M sodium carbonate | 2.5 ml |
| Folin phenol reagent | 0.5 ml |
9. Mix da kyau kafin incubating a cikin ruwa wanka a 37 ℃ na 30 mins.
10. OD660an ƙaddara shi azaman OD1;Ƙungiyar kula da blank: Ana amfani da diluent Enzyme don maye gurbin maganin enzyme don ƙayyade OD660kamar OD2, ΔOD=OD1- OD2.
11. L-tyrosine misali kwana: 0.5mL daban-daban maida hankali L-tyrosine bayani, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent a 5mL centrifuge tube, incubate a 37 ℃ for 30mins, gano ga OD660don daban-daban maida hankali na L-tyrosine, sa'an nan samu daidaitaccen kwana Y = kX + b, inda Y ne L-tyrosine maida hankali, X ne OD.600.
Lissafi
2: Jimlar yawan maganin amsawa (mL)
0.5: Girman maganin enzyme (mL)
0.5: Ƙarar ruwa mai amsawa da aka yi amfani da shi a cikin ƙaddarar chromogenic (mL)
10: Lokacin amsawa (minti)
Df: Dilution da yawa
CEnzyme maida hankali (mg/ml)
Figures
Hoto 1 ragowar DNA
| Misali | Ave C4 | Nucleic acid Farfadowa(pg/mg) | Farfadowa(%) | Jimlar 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-H2O | Ba a tantance ba | - | - | - |
Hoto 2 Mafi kyawun pH
Hoto 3 Mafi kyawun zafin jiki
Hoto 4 pH Tsayar da hankali
Hoto 5 Zaman lafiyar thermal
Hoto 6 Kwanciyar hankali a 25 ℃
