ʻO Proteinase K (Ka wai)
Helu popoki: HC4502A
ʻO ka Proteinase K kahi protease serine kūpaʻa me ka kikoʻī o ka substrate ākea.Hoʻohaʻahaʻa ia i nā protein he nui i ka mokuʻāina maoli a i mua o nā mea holoi.Hōʻike nā hōʻike mai nā haʻawina aniani a me ka molecular structure e hōʻike ana i ka enzyme no ka ʻohana subtilisin me kahi kahua catalytic triad (Asp).39-ʻO kāna69- Ser224).ʻO ka pae nui o ka cleavage ʻo ia ka paʻa peptide e pili ana i ka hui carboxyl o aliphatic a me nā ʻakika amino ʻala me nā pūʻulu alpha amino i hoʻopaʻa ʻia.Hoʻohana mau ʻia no kona ākeakikoʻī.
Hōʻike
| Ka nana aku | Wai waihoʻoluʻu a ʻeleʻele māmā |
| Ka hana | ≥800 U/ml |
| ʻO ka hoʻopaʻa ʻana i ka protein | ≥20 mg/ml |
| DNase | ʻAʻohe mea i ʻike ʻia |
| RNase | ʻAʻohe mea i ʻike ʻia |
Nā Kūlana Waihona
E mālama i ka mahana o 2-8 ℃.
Waiwai
| Helu EC | 3.4.21.64 (Rhui pū ʻia mai ka album Tritirachium) |
| Kaumaha molekula | 29 kDa (SDS-PAGE) |
| kiko isoelectric | 7.81 |
| pH maikaʻi loa | 7.0-12.0 Fig.1 |
| ʻO ka wela kūpono | 65 ℃ Fig.2 |
| paʻa pH | pH 4.5-12.5 (25 ℃, 16 h) Fig.3 |
| Paʻa wela | Ma lalo o 50 ℃ (pH 8.0, 30 min) Fig.4 |
| Nā mea hoʻoikaika | SDS, urea |
| Nā mea pale | Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Nā noi
1. Puke diagnostic genetic
2. RNA a me DNA pahu pahu
3. ʻO ka unuhi ʻana i nā ʻāpana non-protein mai nā ʻiʻo, ka hoʻohaʻahaʻa ʻana i nā haumia protein, e like me nā kano DNA a me ka hoʻomākaukau ʻana i ka heparin
4. Ka hoʻomākaukau ʻana o ka chromosome DNA ma ka pulsed electrophoresis
5. Pili komohana
6. Enzymatic glycosylated albumin reagents in vitro diagnostics
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.
Hoʻāʻo
Wehewehe ʻāpana
Hoʻokahi ʻāpana (U) i wehewehe ʻia e like me ka nui o ka enzyme e koi ʻia e hydrolyze casein e hana ai i 1 μmol tyrosine i kēlā me kēia minuke ma lalo o kēia mau kūlana.
Hoʻomākaukau ʻana i nā reagents
Reagent I: 1g waiu casein ua hoʻoheheʻe ʻia i loko o 50ml o 0.1M sodium phosphate solution (pH 8.0), incubated i loko o 65-70 ℃ wai no 15mins, hoʻoulu ʻia a hoʻoheheʻe ʻia, hoʻoluʻu ʻia e ka wai, hoʻoponopono ʻia e ka sodium hydroxide i pH8.0, a paʻa. ka nui 100ml.
Reagent II: Hoʻonā TCA: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
Reagent III: 0.4M Na2CO3hoʻonā.
Reagent IV: Forint reagent i hoʻoheheʻe ʻia me ka wai maʻemaʻe no 5 mau manawa.
Reagent V: Mea hoʻoheheʻe Enzyme: 0.1M solution sodium phosphate (pH 8.0).
Reagent VI: Hoʻonā Tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine i hoʻoheheʻe ʻia me 0.2M HCl.
Kaʻina hana
1. 0.5ml o ka reagent I ua hoʻomaʻamaʻa muaʻia i ka 37 ℃, e hoʻohui i ka 0.5ml o ka enzyme solution, e hui maikaʻi, a hoʻoulu i 37 ℃ no 10mins.
2. Hoʻohui i 1ml o ka reagent II e hoʻopau i ka hopena, hui maikaʻi, a hoʻomau i ka incubation no 30mins.
3. Centrifugate hoʻonā hopena.
4. E lawe i ka supernatant 0.5ml, e hoʻohui i ka 2.5ml reagent III, 0.5ml reagent IV, hui maikaʻi a hoʻomoʻa i 37 ℃ no 30mins.
5. OD660Ua hoʻoholo ʻia ʻo OD1;pūʻulu mana blank: 0.5ml reagent V hoʻohana ʻia e pani i ka hopena enzyme e hoʻoholo ai i ka OD660e like me OD2, ΔOD=OD1-OD2.
6. L-tyrosine ka pihi maʻamau: 0.5mL ʻokoʻa ka manaʻo L-tyrosine solution, 2.5mL Reagent III, 0.5mL Reagent IV 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 kuhikuhi
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.Biophys.Res.Commun.(1971);44:513.
3. Hilz, H.et al.,Eur.J. Biochem.(1975);56:103–108.
4. Sambrook Jet al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(1989).
Nā huahelu
Fig. 1 ʻOi loa pH
100mM hoʻonā pale: pH6.0-8.0, Na-phosphate;pH8.0- 9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Enzyme hoʻopalekana:1mg/mL
Fig. 2 ʻO ka wela kūpono
ʻO ka pane ʻana ma 20mM K-phosphate buffer pH 8.0.ʻO ka hoʻopaʻa ʻana o ka enzyme: 1mg/mL
Fig. 3 pH Paʻa
25 ℃, 16 h-hana me ka 50mM buffer solution: 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.ʻO ka hoʻopaʻa ʻana o ka enzyme: 1mg/mL
Fig. 4 Thermal kūpaʻa
30 mau minuke me ka 50mM Tris-HCl buffer, pH 8.0.ʻO ka hoʻopaʻa ʻana o ka enzyme: 1mg/mL
Fig. 5 Waihona kūpaʻaty at 25 ℃
