Imikhiqizo

Usawoti we-Tris acetate uvame ukusetshenziswa ekulungiseni i-TAE (Tris-acetate-EDTA) buffer, esetshenziswa njengesigcinamandla esisebenzayo kanye namajeli e-agarose.Ibhafa ye-Tris Acetate-EDTA isetshenziselwa i-DNA agarose gel electrophoresis kodwa futhi isetshenziselwa i-RNA agarose gel electrophoresis engakhiqizi i-denaturing.I-DNA enemicu ekabili ijwayele ukusebenza ngokushesha ku-TAE kunakwezinye izigcinalwazi futhi ingase ikhathale ngesikhathi se-electrophoresis enwetshiwe.Ukujikeleza kwebhafa noma ukushintshwa kwebhafa ngesikhathi se-electrophoresis enwetshiwe kungalungisa umthamo ophansi wokubhafa.Ingasetshenziswa ezindaweni ezihlukahlukene ukutadisha ukuhamba kwe-DNA ekuxazululeni.Njengoba i-borate kubhafa ye-TBE (i-Tris-Borate-EDTA Buffer, 10X Powder Pack, sc-296651) iyisivimbeli esiqinile sama-enzyme amaningi, isigcinalwazi se-TAE siyanconywa uma ubheka izinhlelo zokusebenza ze-enzymatic zesampula ye-DNA.Usawoti we-Tris acetate futhi uyisivikelo esizwela kakhulu ekuhlolweni kwe-ATP nge-firefly luciferase.

Ukusetshenziswa: I-TAE egijima ibuffer iyibhafa evame ukusetshenziswa kakhulu ye-DNA agarose gel electrophoresis, futhi iphinde isetshenziselwe i-RNA agarose gel electrophoresis yomdabu.I-DNA enemicu ekabili ijwayele ukuhamba ngokushesha ku-TAE kunakwezinye izigcinalwazi, kodwa futhi yehluleka ukubhukuda ngenxa yokuncipha kwama-ion e-buffer ngesikhathi se-electrophoresis ende.Ukuhamba ngebhafa ngamabhayisikili noma ukushintsha kwebhafa ngesikhathi se-electrophoresis ende kunganxephezela umthamo ophansi webhafa.2 Nciphisa ibhafa ye-TAE egxilile ukuze uthole i-1 mMTAE buffer equkethe 40 mM Tris acetate kanye no-1 mM EDTA, pH 8.3.I-1 mMTAE buffer ingasetshenziswa kokubili kumajeli e-agarose nanjengesigcinalwazi esisebenzayo.Ukuze uthole ukulungiswa okuphezulu, kunconywa ukuthi i-voltage esetshenzisiwe ibe ngaphansi kuka-5V/cm (ibanga phakathi kwama-electrode amayunithi).

Isicelo: I-TAE egijima ibuffer iyindawo evame ukusetshenziswa kakhulu ye-DNA agarose gel electrophoresis kujeli ye-Chemicalbook, futhi iphinde isetshenziselwe i-RNA agarose gel electrophoresis engakhiqizi i-denaturing.I-DNA enemicu ekabili ijwayele ukuhamba ngokushesha ku-TAE kunakwezinye izigcinalwazi, kodwa futhi yehluleka ukubhukuda ngenxa yokuncipha kwama-ion e-buffer ngesikhathi se-electrophoresis ende.Ukuhamba ngebhafa ngamabhayisikili noma ukushintsha kwebhafa ngesikhathi se-electrophoresis ende kunganxephezela umthamo ophansi webhafa.Ibhafa ye-TAE egxilisiwe yahlanjululwa ukuze kutholwe isibhafa esingu-1 mMTAE esiqukethe 40 mM Tris acetate kanye no-1 mM EDTA, pH 8.3.I-1 mMTAE buffer ingasetshenziswa kokubili kumajeli e-agarose nanjengesigcinalwazi esisebenzayo.Ukuze uthole ukulungiswa okuphezulu, kunconywa ukuthi i-voltage esetshenzisiwe ibe ngaphansi kuka-5V/cm (ibanga phakathi kwama-electrode amayunithi).

Ukusetshenziswa: Ekutholweni kwe-ATP nge-firefly luciferase, lo mkhiqizo uyisivikelo esizwela kakhulu;ukutholwa kokubopha kwe-glutamate.

Ukubophezela okususwayo kwe-[3H]l-glutamic acid ezintweni ezingamukeli.

[3H]I-L-glutamic acid ebophezela kumashubhu e-microfuge nengilazi iye yaphenywa kumabhafa amane.Ukubophezela okungemuva kulezi zinto zokusebenza bekunganaki, kodwa kunyuswe ngokufakwa phakathi nendawo noma ukumuncwa ku-Tris-HCl ne-Tris-citrate buffer.Lokhu kubophezela bekukuncane kakhulu noma Lapho i-HEPES-KOH, noma i-Tris-acetate buffer isetshenziswa esikhundleni salokho.[3H]I-L-glutamate ebophezela kumashubhu e-microfuge iye yavinjwa ama-L- kodwa hhayi ama-D-isomers we-glutamate ne-aspartate.I-DL-2-amino-7-phosphonoheptanoic acid nayo ayizange ivimbele ukubopha.Amanye ama-compounds abonise ukuvinjelwa okuphansi kuya kokulinganisela kwakuyi-N-methyl-D-aspartate, i-quisqualate, i-L-glutamic acid diethyl ester, i-N-methyl-L-aspartate, i-kainate, ne-2-amino-4 -phosphonobutyrate.Ukubophezela kuye kwavinjwa ulwelwesi lobuchopho begundane.Ukubophezela kwe-glutamate okuncike kumaprotheni [3H] kutholwe ngokulungiswa kolwelwesi oluncibilikisiwe oluqandiswe ngokuphindaphindiwe lapho ukubopha kwenziwa kubhafa ye-Tris-acetate.Kunconywa ukuthi i-Tris-acetate noma i-HEPES -KOH buffer kufanele isetshenziswe ku-glutamate bin ding assay.Uma i-Tris-HCl noma i-Tris-citrate buffer isetshenziswa, ukuhlolwa kokulawula okufanele kufanele kwenziwe ukuze kulungiswe ukubophezela kumashubhu e-microfuge noma izihlungi ze-fiber zengilazi.