Iimveliso

Ityuwa ye-acetate ye-Tris isetyenziswa rhoqo ekulungiseleleni i-TAE (Tris-acetate-EDTA) buffer, esetyenziswa njengesithinteli esisebenzayo kunye neejeli ze-agarose.I-Tris Acetate-EDTA buffer isetyenziselwa i-DNA agarose gel electrophoresis kodwa ikwasetyenziswa kwi-non-denaturing RNA agarose gel electrophoresis.I-DNA ephindwe kabini ithande ukubaleka ngokukhawuleza kwi-TAE kunezinye izithinteli kwaye inokudinwa ngexesha elongezelelweyo le-electrophoresis.Ukujikeleziswa kwe-buffer okanye ukutshintshwa kwe-buffer ngexesha elongeziweyo le-electrophoresis kunokulungisa umthamo osezantsi wobufa.Isenokusetyenziswa kwiindawo ezahlukeneyo ukufunda ukushukuma kwe-DNA kwisisombululo.Ekubeni i-borate kwi-TBE buffer (i-Tris-Borate-EDTA Buffer, i-10X Powder Pack, sc-296651) isithinteli esinamandla kwii-enzymes ezininzi, i-TAE buffer iyacetyiswa xa ujonga izicelo ze-enzymatic zesampuli ye-DNA.Ityuwa ye-acetate ye-Tris ikwasisithinteli esinovakalelo oluphezulu kwiimvavanyo ze-ATP ngefirefly luciferase.

Usetyenziso: I-TAE eqhuba isithinteli sesona sithinteli sisetyenziswa kakhulu kwi-DNA agarose gel electrophoresis, kwaye sikwasetyenziselwa i-RNA agarose gel electrophoresis.I-DNA ephindwe kabini ithande ukuhamba ngokukhawuleza kwi-TAE kunezinye izithinteli, kodwa ikwasilela ukuqubha ngenxa yokuncipha kwee-ion ze-buffer ngexesha le-electrophoresis ende.Ukuhamba ngebhayisikile yesithinteli okanye ukutshintshiselana ngesithinteli ngexesha le-electrophoresis ende kunokubuyekeza umthamo ophantsi wesithinteli.2 Dibanisa isithinteli esigxininisiweyo se-TAE ukufumana i-1 mMTAE buffer equlethe i-40 mM Tris acetate kunye ne-1 mM EDTA, pH 8.3.I-1 mMTAE buffer ingasetyenziswa zombini kwiijeli ze-agarose nanjengesithinteli esibalekayo.Ukufumana isisombululo esiphezulu, kucetyiswa ukuba i-voltage esetyenzisiweyo ibe ngaphantsi kwe-5V / cm (umgama phakathi kweeyunithi ze-electrodes).

Isicelo: I-TAE eqhuba isithinteli sesona sixhasi sixhaphakileyo sisetyenziswa kwi-DNA agarose gel electrophoresis kwi-Chemicalbook gel, kwaye ikwasetyenziselwa non-denaturing RNA agarose gel electrophoresis.I-DNA ephindwe kabini ithande ukuhamba ngokukhawuleza kwi-TAE kunezinye izithinteli, kodwa ikwasilela ukuqubha ngenxa yokuncipha kwee-ion ze-buffer ngexesha le-electrophoresis ende.Ukuhamba ngebhayisikile yesithinteli okanye ukutshintshiselana ngesithinteli ngexesha le-electrophoresis ende kunokubuyekeza umthamo ophantsi wesithinteli.Isithinteli esigxininisiweyo se-TAE saxutywa ukufumana i-1 mMTAE isithinteli esiqulethe i-40 mM Tris acetate kunye ne-1 mM EDTA, pH 8.3.I-1 mMTAE buffer ingasetyenziswa zombini kwiijeli ze-agarose nanjengesithinteli esibalekayo.Ukufumana isisombululo esiphezulu, kucetyiswa ukuba i-voltage esetyenzisiweyo ibe ngaphantsi kwe-5V / cm (umgama phakathi kweeyunithi ze-electrodes).

Isebenzisa: Ekubhaqweni kwe-ATP nge-firefly luciferase, le mveliso yeyona nto inobungozi;ukubhaqwa kokubophelela kwe-glutamate.

Ukubophelela okushenxiswayo kwe- [3H] l-glutamic acid kwizinto ezingezo-receptor.

[3H]L-glutamic acid ebophelelayo kwiityhubhu ze-microfuge kunye neglasi yaphandwa kwizithinteli ezine.Imvelaphi yokubophelela kwezi zixhobo yayingenamsebenzi, kodwa yonyuswa nge-centrifugation okanye ukufunxa kwi-Tris-HCl kunye ne-Tris-citrate buffer.Oku kubophelela bekungaphantsi kakhulu okanye Xa i-HEPES-KOH, okanye i-Tris-acetate buffer isetyenzisiwe endaweni yoko.[3H]I-L-glutamate ebophelelayo kwiityhubhu ze-microfuge yayithintelwe yi-L- kodwa kungekhona i-D-isomers ye-glutamate kunye ne-aspartate.I-DL-2-amino-7-phosphonoheptanoic acid nayo ayizange ivimbele ukubopha.Ezinye iikhompawundi ezibonise inhibition ephantsi ukuya kwimodareyitha yayiyi: N-methyl-D-aspartate, quisqualate, L-glutamic acid diethyl ester, N-methyl-L-aspartate, kainate, kunye ne-2-amino-4 -phosphonobutyrate.Ukuzibophelela kwakuthintelwe ziinwebu zobuchopho zempuku.Isibophelelo esixhomekeke kwiprotheni [3H] glutamate sifunyenwe ngokulungiswa kwenwebu ekhenkcezayo ekhenkcezisiweyo xa ukubophelela kwakusenziwa kwi-Tris-acetate buffer.Kucetyiswa ukuba i-Tris-acetate okanye i-HEPES -KOH buffer kufuneka isetyenziswe kwi-glutamate bin ding assay.Ukuba i-Tris-HCl okanye i-Tris-citrate buffer isetyenzisiwe, umfuniselo wolawulo olufanelekileyo kufuneka lwenziwe ukulungisa ukubophelela kwiityhubhu ze-microfuge okanye izihluzi zefiber zeglasi.