Proteinase K NGS (pauka)
Helu popoki: HC4507A
ʻO NGS Protease K he protease serine paʻa me ka hana enzyme kiʻekiʻe a me ka kikoʻī substrate ākea. ʻO ka enzyme makemake e hoʻopau i nā paʻa ester a me nā paʻa peptide e pili ana i ka C-terminal o nā waikawa amino hydrophobic, sulfur-containing amino acids a aromatic amino acids.No laila, hoʻohana pinepine ʻia e hoʻohaʻahaʻa i nā protein i nā peptides pōkole.ʻO NGS Protease K he protease serine maʻamau me ka Asp39-ʻO kāna69-Ser224catalytic triad he mea kū hoʻokahi i nā protease serine, a ua hoʻopuni ʻia ke kikowaena catalytic e ka tow Ca.2+nā wahi paʻa no ka hoʻopaʻa ʻana, mālama i ka hana enzyme kiʻekiʻe ma lalo o kahi ākea ākea.
Hōʻike
| Ka nana aku | ʻO ka pauka amorphous keʻokeʻo a keʻokeʻo, lyophilized |
| Hana kikoʻī | ≥40U/mg paʻa |
| DNase | ʻAʻohe mea i ʻike ʻia |
| RNase | ʻAʻohe mea i ʻike ʻia |
| ʻO ke kaumaha kino | ≤50CFU/g paʻa |
| Ke koena nucleic acid | <5pg/mg paʻa |
Waiwai
| Puna | Puke mele Tritirachium |
| Helu EC | 3.4.21.64(Recombinant mai ka album Tritirachium) |
| Kaumaha molekula | 29kDa (SDS-PAGE) |
| kiko isoelectric | 7.81 Fig.1 |
| pH maikaʻi loa | 7.0-12.0 (Hana nā mea a pau i ka hana kiʻekiʻe) Fig.2 |
| ʻO ka wela kūpono | 65 ℃ Fig.3 |
| pH Paʻa | pH 4.5-12.5 (25℃,16h) Fig.4 |
| Paʻa wela | Ma lalo o 50 ℃ (pH 8.0, 30min) Fig.5 |
| Paʻa mālama | Mālama ʻia ma 25 ℃ no 12 mau mahina Fig.6 |
| Nā mea hoʻoikaika | SDS, urea |
| Nā mea pale | Diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Nā Kūlana Waihona
E mālama i ka pauka lyophilized ma -25 ~ -15 ℃ no ka manawa lōʻihi mai ka mālamalama;Ma hope o ka hoʻoheheʻe ʻana, e hoʻokaʻawale i ka leo kūpono no ka mālama pōkole ma 2-8 ℃ mai ka mālamalama a i ʻole mālama lōʻihi ma -25 ~ -15 ℃ mai ka mālamalama.
Hoʻomalu
E hoʻohana i nā mīkina lima pale a me nā maka aniani i ka wā e hoʻohana ai a i ke kaupaona ʻana, a mālama pono i ka ea ma hope o ka hoʻohana ʻana.Hiki i kēia huahana ke hoʻoulu i ka maʻi ʻili a me ka ʻeha nui o ka maka.Inā inhaled, hiki ke hoʻoulu i ka maʻi a me nā hōʻailona hānō a i ʻole dyspnea.Hiki ke hoʻopilikia i ka hanu.
Wehewehe ʻāpana
Hoʻokahi ʻāpana o NGS Protease K i wehewehe ʻia e like me ka nui o ka enzyme i koi ʻia e hydrolyze casein i loko o 1 μmol L-tyrosine ma lalo o nā kūlana hoʻoholo maʻamau.
Hoʻomākaukau ʻana i nā reagents
| Reagent | Mea hana | Heluhelu |
| ʻenehana Caseinmai ka waiu bipi | Sigma Aldrich | C7078 |
| NaOH | ʻO Sinopharm ChemicalReagent Co., Ltd. | 10019762 |
| NaH2PO4·2H2O | ʻO Sinopharm ChemicalReagent Co., Ltd. | 20040718 |
| Na2HPO4 | ʻO Sinopharm ChemicalReagent Co., Ltd. | 20040618 |
| ʻAka Trichloroacetic | ʻO Sinopharm ChemicalReagent Co., Ltd. | 80132618 |
| Sodium acetate | ʻO Sinopharm ChemicalReagent Co., Ltd. | 10018818 |
| Acetic acid | ʻO Sinopharm ChemicalReagent Co., Ltd. | 10000218 |
| HCl | ʻO Sinopharm ChemicalReagent Co., Ltd. | 10011018 |
| ʻO ka sodium carbonate | ʻO Sinopharm ChemicalReagent Co., Ltd. | 10019260 |
| Foline-phenol | Sangon Biotech (Shanghai)Co., Ltd. | A500467-0100 |
| L-tyrosine | Sigma | 93829 |
Reagent I:
Lapaʻa: 1% Casein mai ka waiu waiu bipi: hoʻoheheʻe i 1g bovine milk casein i 50ml o 0.1M sodium phosphate solution, pH 8.0, wela i ka wai ʻauʻau ma 65-70 °C no 15mins, hoʻoulu a hoʻoheheʻe, hoʻoluʻu me ka wai, hoʻoponopono ʻia e ka sodium hydroxide i ka pH 8.0, a hoʻoheheʻe i ka 100ml.
Luna Hoʻoponopono II:
TCA solution: 0.1M trichloroacetic acid, 0.2M sodium acetate a me 0.3M acetic acid (kaumaha 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid successively, hoʻohui i 50mL deionized wai, hoʻoponopono me HCl3 i loko o ka HCl3. 100ml).
Luna Hoʻoponopono III:
0.4m sodium carbonate solution (kaumaha 4.24g anhydrous sodium carbonate a hoʻoheheʻe i loko o 100mL wai)
Luna Hoʻoponopono IV:
Folin phenol reagent: dilute 5 manawa me ka wai deionized.
Luna Hoʻopaʻa V:
Mea hoʻoheheʻe enzyme: 0.1 M ka hopena sodium phosphate, pH 8.0.
Luna Hoʻopaʻa VI:
L-tyrosine hoʻonā maʻamau: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml L-tyrosine i hoʻoheheʻe ʻia me 0.2M HCl.
Kaʻina hana
1. E ho'ā i ka spectrophotometer UV-Vis a koho i ke ana photometric.
2. E hoʻonoho i ka nalu nalu e like me 660nm.
3. E hoʻohuli i ka ʻauʻau wai, e hoʻonoho i ka mahana i 37 ℃, e hōʻoia i ka loli ʻole o ka mahana no 3-5mins.
4. E hoʻomaʻamaʻa mua i ka substrate 0.5mL i loko o kahi paipu centrifuge 2mL ma 37 ℃ ʻauʻau wai no 10mins.
5. E unuhi i 0.5mL i hoʻoheheʻe ʻia i loko o ka pahu centrifuge preheated no 10 mau minuke.E hoʻonoho i ka diluent enzyme ma ke ʻano he pūʻulu hakahaka.
6. E hoʻohui i 1.0 mL TCA reagent ma hope koke o ka hopena.E hui maikaʻi a hoʻokomo i loko o ka ʻauʻau wai no 30 mau minuke.
7. Centrifugate hoʻonā hopena.
8. E hoʻohui i kēia mau ʻāpana i ke kauoha i kuhikuhi ʻia.
| Reagent | Volume |
| ʻO ka supernatant | 0.5 mL |
| 0.4M Sodium carbonate | 2.5 mL |
| Folin phenol reagent | 0.5 mL |
9. E hui maikaʻi ma mua o ka hoʻokomo ʻana i ka ʻauʻau wai i 37 ℃ no 30 min.
10. OD660Ua hoʻoholo ʻia ʻo OD1;pūʻulu mana blank: Hoʻohana ʻia ka diluent enzyme e pani i ka hopena enzyme e hoʻoholo ai i ka OD660e like me OD2, ΔOD=OD1-OD2.
11. L-tyrosine ka pihi maʻamau: 0.5mL okoa ka manaʻo L-tyrosine solution, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent i 5mL centrifuge tube, incubate i 37 ℃ no 30mins, ʻike no OD660no ka ʻokoʻa o ka hoʻopaʻa ʻana o L-tyrosine, a laila loaʻa ka ʻōkuhi maʻamau Y=kX+b, kahi ʻo Y ka manaʻo L-tyrosine, X ʻo OD.600.
Heluhelu
2: Ka nui o ka nui o ka hoʻonā hopena (mL)
0.5: Ka nui o ka hopena enzyme (mL)
0.5: Hoʻohana ʻia ka nui o ka wai i ka hoʻoholo chromogenic (mL)
10: Manawa pane (min)
Df: Dilution lehulehu
C: Ke kuʻekuʻe o ka enzyme (mg/mL)
Nā huahelu
Fig.1 DNA koena
| Laʻana | Ave C4 | Nucleic Acid Hoʻihoʻi (pg/mg) | Hoʻihoʻi (%) | Huina Nucleic ʻakika ( 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 | ʻAʻole maopopo | — | — | — |
Fig.2 pH maikaʻi loa
Fig.3 ʻO ka wela kūpono
Fig.4 pH Paʻa
Fig.5 Paʻa wela
Fig.6 Paʻa mālama ma 25 ℃
