Proteinase K NGS (ntụ ntụ)
Nọmba nwamba: HC4507A
NGS Protease K bụ serine protease kwụsiri ike nke nwere ọrụ enzyme dị elu yana ụdị mkpụrụ dị obosara.Ya mere, a na-ejikarị ya emebi protein n'ime obere peptides.NGS Protease K bụ ụdị protease serine nwere Asp39-Ya69-Ser224catalytic triad nke pụrụ iche na serine proteases, na catalytic center gbara ya gburugburu tow Ca.2+saịtị njikọ maka nkwụsi ike, na-ejigide ọrụ enzyme dị elu n'okpuru ọnọdụ dịgasị iche iche.
Nkọwapụta
| Ọdịdị | Amorphous ntụ ntụ na-acha ọcha ruo na-acha ọcha, lyophilized |
| Ọrụ akọwapụtara | ≥40U/mg siri ike |
| DNA | Ọnweghị nke achọpụtara |
| RNase | Ọnweghị nke achọpụtara |
| Ibu ibu | ≤50CFU/g siri ike |
| Nucleic acid fọdụrụnụ | <5pg/mg siri ike |
Njirimara
| Isi mmalite | Tritirachium album |
| Nọmba EC | 3.4.21.64(Mgbakọ sitere na Tritirachium album) |
| Ibu molekụla | 29kDa (SDS-PAGE) |
| Isi ihe nke isoelectric | 7.81 Foto.1 |
| pH kacha mma | 7.0-12.0 (Ha niile na-arụ ọrụ dị elu) Fig.2 |
| Okpomọkụ kacha mma | 65℃ Fig.3 |
| pH kwụsie ike | pH 4.5-12.5 (25 ℃, 16h) Fig.4 |
| Nkwụsi ike okpomọkụ | N'okpuru 50 ℃ (pH 8.0, 30min) Fig.5 |
| Nkwụsi ike nchekwa | Echekwara na 25℃ maka ọnwa 12 Fig.6 |
| Ndị na-eme ihe | SDS, urea |
| Ihe mgbochi | Diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Ọnọdụ Nchekwa
Chekwaa ntụ ntụ lyophilized na -25 ~ -15 ℃ ruo ogologo oge pụọ na ìhè;Mgbe mgbasa, tinye n'ime olu kwesịrị ekwesị maka nchekwa oge dị mkpirikpi na 2-8 ℃ pụọ na ìhè ma ọ bụ nchekwa ogologo oge na -25 ~ -15 ℃ pụọ na ìhè.
Mkpachapụ anya
Yiri gloves nchebe na enyo mgbe ị na-eji ma ọ bụ na-atụ aro, ma na-ekuku ikuku nke ọma mgbe ejiri ya.Ngwaahịa a nwere ike ibute mmeghachi ahụ nfụkasị anụ ahụ yana oke iwe anya.Ọ bụrụ na ikuru ume, ọ nwere ike ibute nfụkasị ahụ ma ọ bụ mgbaàmà ụkwara ume ọkụ ma ọ bụ dyspnea.Nwere ike ịkpata mgbakasị iku ume.
Nkọwa nkeji
A kọwapụtara otu nkeji NGS Protease K dị ka ọnụọgụ enzyme achọrọ iji hydrolyze casein n'ime 1 μmol L-tyrosine n'okpuru ọnọdụ mkpebi ọkọlọtọ.
Reagents nkwadebe
| Reagent | Onye nrụpụta | Katalọgụ |
| Ozi gbasara ụlọ ọrụ Caseinsite na mmiri ara ehi | Sigma Aldrich | C7078 |
| NaOH | Sinopharm ChemicalỤgwọ nke ụlọ ọrụ Reagent Co., Ltd. | 10019762 |
| NaH2PO4· 2H2O | Sinopharm ChemicalỤgwọ nke ụlọ ọrụ Reagent Co., Ltd. | 20040718 |
| Nke 2HPO4 | Sinopharm ChemicalỤgwọ nke ụlọ ọrụ Reagent Co., Ltd. | 20040618 |
| Trichloroacetic acid | Sinopharm ChemicalỤgwọ nke ụlọ ọrụ Reagent Co., Ltd. | 80132618 |
| Sodium acetate | Sinopharm ChemicalỤgwọ nke ụlọ ọrụ Reagent Co., Ltd. | 10018818 |
| Acetic acid | Sinopharm ChemicalỤgwọ nke ụlọ ọrụ Reagent Co., Ltd. | 10000218 |
| HCl | Sinopharm ChemicalỤgwọ nke ụlọ ọrụ Reagent Co., Ltd. | 10011018 |
| Sodium carbonate | Sinopharm ChemicalỤgwọ nke ụlọ ọrụ Reagent Co., Ltd. | 10019260 |
| Foline-phenol | Sangon Biotech (Shanghai)Ụlọ ọrụ, Ltd. | A500467-0100 |
| L-tyrosine | Sigma | 93829 |
Reagent I:
Mkpụrụ: 1% Casein si mmiri ara ehi mmiri ara ehi: igbari 1g bovine mmiri ara ehi casein na 50ml nke 0.1M sodium phosphate solution, pH 8.0, okpomọkụ na mmiri bat na 65-70 Celsius C maka 15mins, bilie na igbari, dị jụụ na mmiri, gbanwee site na. sodium hydroxide ruo pH 8.0, na itughari na 100ml.
Reagent II:
Ngwọta TCA: 0.1M trichloroacetic acid, 0.2M sodium acetate na 0.3M acetic acid (dị 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid n'otu n'otu, tinye 50mL deionized mmiri, gbanwee na HCl na p.p.3H. 100 ml).
Reagent III:
0.4m sodium carbonate ngwọta (tuo 4.24g anhydrous sodium carbonate na igbari na 100ml mmiri)
Reagent IV:
Folin phenol reagent: jiri mmiri deionized gbanye ugboro 5.
Reagent V:
Enzyme diluent: 0.1 M sodium phosphate ngwọta, pH 8.0.
Reagent VI:
Ngwọta ọkọlọtọ L-tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml L-tyrosine gbazere na 0.2M HCl.
Usoro
1. Gbanwuo UV-Vis spectrophotometer wee họrọ nha fotometric.
2. Tọọ ogologo ikuku ka 660nm.
3. Gbanyụọ bat mmiri, mee ka okpomọkụ dị na 37 ℃, hụ na okpomọkụ adịghị agbanwe maka 3-5mins.
4. Preheat 0.5mL mkpụrụ na a 2mL centrifuge tube na 37 ℃ mmiri saa maka 10mins.
5. Wepụ 0.5mL ngwọta enzyme diluted n'ime tube centrifuge preheated maka nkeji 10.Tọọ ihe mgbaze enzyme dị ka otu oghere.
6. Tinye 1.0 mL TCA reagent ozugbo mmeghachi omume ahụ gasịrị.Gwakọta nke ọma ma tinye ya na mmiri ịsa mmiri maka nkeji iri atọ.
7. Centrifugate mmeghachi omume ngwọta.
8. Tinye ihe ndị a n'usoro akọwapụtara.
| Reagent | Olu |
| Supernatant | 0,5 ml |
| 0.4M sodium carbonate | 2.5 ml |
| Folin phenol reagent | 0,5 ml |
9. Mix ọma tupu incubating na mmiri bat a 37 ℃ maka 30 mins.
10. OD660e kpebisiri ike dị ka OD1;Otu njikwa oghere: A na-eji diluent enzyme dochie ngwọta enzyme iji chọpụta OD660dị ka OD2, ΔOD=OD1-Od2.
11. L-tyrosine ọkọlọtọ usoro: 0.5mL iche iche ịta L-tyrosine ngwọta, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent na 5mL centrifuge tube, incubate na 37 ℃ maka 30mins, chọpụta maka OD660maka ntinye uche dị iche iche nke L-tyrosine, wee nweta usoro ọkọlọtọ Y = kX + b, ebe Y bụ ntinye L-tyrosine, X bụ OD.600.
Mgbakọ
2: ngụkọta olu ngwọta mmeghachi omume (mL)
0.5: Olu nke ngwọta enzyme (mL)
0.5: Mmeghachi omume olu mmiri mmiri ejiri na mkpebi chromogenic (mL)
10: Oge mmeghachi omume (nkeji)
Df: Dilution otutu
CNtinye nke enzymes (mg/mL)
Ọnụọgụ
Fig.1 ihe fọdụrụ DNA
| Ihe atụ | Ụzọ C4 | Nucleic acid mgbake (pg/mg) | mgbake(%) | Mgbakọta 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 efu | Ekpebibeghị | - | - | - |
Fig.2 pH kacha mma
Fig.3 okpomọkụ kacha mma
Fig.4 pH Nkwụsi ike
Fig.5 Nkwụsi ike nke okpomọkụ
Fig.6 Nkwụsi ike nchekwa na 25 ℃
