Proteinase K mNGS (ruwa)
Proteinase K shine bargaren serine protease tare da faffadan keɓantacce.Yana ƙasƙantar da sunadaran da yawa a cikin asalin ƙasar ko da a gaban abubuwan wanke-wanke.Shaida daga binciken tsarin kristal da kwayoyin halitta sun nuna cewa enzyme na cikin dangin subtilisin ne tare da triad na catalytic site (Asp).39-Nasa69-Ser224).Babban wurin cleavage shine haɗin peptide kusa da rukunin carboxyl na aliphatic da amino acid masu kamshi tare da toshe ƙungiyoyin alfa amino.An fi amfani da shi don ƙayyadaddun ƙayyadaddun sa.Wannan furotin K an tsara shi musamman don mNGS.Idan aka kwatanta da sauran proteinase K, yana ƙunshe da ƙarancin gurɓataccen acid nucleic tare da aikin enzymatic iri ɗaya, wanda zai iya tabbatar da aikace-aikacen mNGS na ƙasa.
Yanayin Ajiya
2-8 ℃ na shekaru 2
Ƙayyadaddun bayanai
| Bayyanar | Ruwa mara launi zuwa launin ruwan kasa |
| Ayyuka | ≥800 U/ml |
| Rarraba Protein | ≥20 mg/ml |
| Nickase | Babu wanda aka gano |
| DNA | Babu wanda aka gano |
| RNase | Babu wanda aka gano |
Kayayyaki
| lambar EC | 3.4.21.64(Recombinant daga Tritirachium album) |
| Isoelectric batu | 7.81 |
| Mafi kyawun pH | 7.0- 12.0 Hoto 1 |
| Mafi kyawun zafin jiki | 65 ℃ Hoto 2 |
| pH kwanciyar hankali | pH 4.5-12.5 (25 ℃, 16 h) Hoto 3 |
| Zaman lafiyar thermal | Kasa ℃ 50 (pH 8.0, 30 min) Hoto 4 |
| kwanciyar hankali na ajiya | Sama da 90% ayyuka na wata 12 a 25 ℃ |
| Masu kunnawa | SDS, urea |
| Masu hanawa | Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Aikace-aikace
1. Kayan gwajin kwayoyin halitta
2. RNA da kayan cirewar DNA
3. Cire abubuwan da ba su da furotin daga kyallen takarda, lalata gurɓataccen furotin, kamar DNA.maganin rigakafi da shirye-shiryen heparin
4. Shiri na chromosome DNA ta pulsed electrophoresis
5. Yammaci
6. Enzymatic glycosylated albumin reagents in vitro ganewar asali
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
An bayyana raka'a ɗaya (U) azaman adadin enzyme da ake buƙata don yin hydrolyze casein don samar da 1 μmoltyrosine a minti daya a karkashin wadannan yanayi.
Reagents shiri
Reagent I: 1g madara casein an narkar da shi a cikin 50ml na 0.1M sodium phosphate bayani (pH 8.0), incubated a cikin 65-70 ℃ ruwa na 15mins, zuga kuma narkar da, sanyaya da ruwa, gyara ta sodium hydroxide zuwa pH 8.0, da kuma kafaffen girma 100 ml.
Reagent II: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
Reagent III: 0.4M Na2CO3mafita.
Reagent IV: Forint reagent an diluted da ruwa mai tsabta har sau 5.
Reagent V: Enzyme diluent: 0.1M sodium phosphate bayani (pH 8.0).
Reagent VI: maganin tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine narkar da 0.2M HCl.
Tsari
1. 0.5ml na reagent I an pre-warmed zuwa 37 ℃, ƙara 0.5ml na enzyme bayani, Mix da kyau, kuma incubate a37 ℃ na minti 10.
2. Ƙara 1ml na reagent II don dakatar da amsawa, haɗuwa da kyau, kuma ci gaba da shiryawa na 30mins.
3. Centrifugate dauki bayani.
4. Ɗauki 0.5ml supernatant, ƙara 2.5ml reagent III, 0.5ml reagent IV, gauraya da kyau kuma a saka a 37 ℃na 30mins.
5. OD660an ƙaddara shi azaman OD1;Ƙungiyar kula da blank: 0.5ml reagent V ana amfani dashi don maye gurbin enzymebayani don ƙayyade OD660kamar OD2, ΔOD=OD1- OD2.
6. L-tyrosine misali kwana: 0.5mL daban-daban maida hankali L-tyrosine bayani, 2.5mL Reagent III, 0.5mL Reagent IV a 5mL centrifuge tube, incubate a 37 ℃ for 30mins, gano ga OD660don daban-daban maida hankali na L-tyrosine, sa'an nan samu daidaitattun lankwasa 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)
Magana
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.Biophys.Res.Jama'a.(1971);44:513.
3. Hilz, H.da al.,Yuro.J. Biochem.(1975);56:103-108.
4. Sambrook Jet al., Cloning Kwayoyin Halitta: Littafin dakin gwaje-gwaje, bugu na biyu, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989).
Figures
Hoto.1 Mafi kyawu pH
100mM buffer bayani: pH6.0-8.0, Na-phosphate;pH8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Enzyme maida hankali: 1mg/ml
Hoto 2 Mafi kyawun zafin jiki
Amsa a cikin 20 mM K-phosphate buffer pH 8.0.Matsakaicin Enzyme: 1 mg/mL
Hoto.3 pH Kwanciyar hankali
25 ℃, 16 h-jiyya tare da 50 mM buffer bayani: pH 4.5- 5.5, Acetate;pH 6.0-8.0, Na-phosphate;pH 8.0-9.0, Tris-HCl.pH 9.0-12.5, Glycine-NaOH.Matsakaicin Enzyme: 1 mg/mL
Hoto 4 Thermal kwanciyar hankali
30 min-jiyya tare da 50 mM Tris-HCl buffer, pH 8.0.Matsakaicin Enzyme: 1 mg/mL
Hoto 5 Adana kwanciyar hankality at 25 ℃
