Ngwaahịa

A na-ejikarị nnu acetate eme ihe na nkwadebe nke TAE (Tris-acetate-EDTA), nke a na-eji dị ka ihe na-agba ọsọ na gels agarose.A na-eji ihe nchekwa Tris Acetate-EDTA maka DNA agarose gel electrophoresis mana a na-ejikwa ya maka RNA agarose gel electrophoresis na-abụghị denaturing.DNA nwere eriri abụọ na-agba ọsọ ọsọ na TAE karịa na nchekwa ndị ọzọ ma nwee ike ịgwụ ike n'oge electrophoresis agbatị.Mgbasa nchekwa ma ọ bụ ndochi ihe nkpuchi n'oge eletrọphoresis agbatịkwuru nwere ike dozie ike ntanye dị ala.Enwere ike iji ya n'ọtụtụ dị iche iche iji mụọ mmegharị nke DNA na ngwọta.Ebe ọ bụ na borate na TBE buffer (Tris-Borate-EDTA Buffer, 10X Powder Pack, sc-296651) bụ ihe mgbochi siri ike maka ọtụtụ enzymes, TAE buffer na-atụ aro mgbe ị na-elele ngwa enzymatic maka ihe nlele DNA.Nnu Tris acetate bụkwa ihe nchekwa nwere mmetụta dị elu na nyocha ATP na firefly luciferase.

Na-eji: TAE na-agba ọsọ buffer bụ ihe a na-ejikarị eme ihe maka DNA agarose gel electrophoresis, a na-ejikwa ya maka RNA agarose gel electrophoresis.DNA nwere eriri abụọ na-aga ngwa ngwa na TAE karịa na ebe nchekwa ndị ọzọ, mana ọ naghịkwa igwu mmiri n'ihi nkwụsị nke ion buffer n'oge electrophoresis ogologo oge.Ịgba ígwè ma ọ bụ ihe nchekwa ihe ngbanwe n'oge electrophoresis ogologo oge nwere ike kwụọ ụgwọ maka ikike nchekwa dị ala.2 Gwakọta ihe nchekwa TAE gbadoro anya iji nweta ihe nchekwa 1 mMTAE nwere 40 mM Tris acetate na 1 mM EDTA, pH 8.3.Enwere ike iji 1 mMTAE buffer ma na agarose gels yana dị ka ihe nchekwa na-agba ọsọ.Maka mkpebi kachasị, a na-atụ aro ka voltaji etinyere n'ọrụ dị ala karịa 5V / cm (ebe dị n'etiti electrodes unit).

Ngwa: ihe nchekwa TAE na-agba ọsọ bụ ihe a na-ejikarị eme ihe maka DNA agarose gel electrophoresis na gel kemịkalụ, a na-ejikwa ya maka RNA agarose gel electrophoresis na-abụghị denaturing.DNA nwere eriri abụọ na-aga ngwa ngwa na TAE karịa na ebe nchekwa ndị ọzọ, mana ọ naghịkwa igwu mmiri n'ihi nkwụsị nke ion buffer n'oge electrophoresis ogologo oge.Ịgba ígwè ma ọ bụ ihe nchekwa ihe ngbanwe n'oge electrophoresis ogologo oge nwere ike kwụọ ụgwọ maka ikike nchekwa dị ala.A gbajiri ihe nchekwa TAE gbadoro anya iji nweta ihe nchekwa 1 mMTAE nwere 40 mM Tris acetate na 1 mM EDTA, pH 8.3.Enwere ike iji 1 mMTAE buffer ma na agarose gels yana dị ka ihe nchekwa na-agba ọsọ.Maka mkpebi kachasị, a na-atụ aro ka voltaji etinyere n'ọrụ dị ala karịa 5V / cm (ebe dị n'etiti electrodes unit).

Na-eji: Na nchọta nke ATP na firefly luciferase, ngwaahịa a bụ ihe nchebe kachasị mma;nchọpụta njikọ glutamate.

Mwepu nke [3H] l-glutamic acid na ihe anaghị anabata ya.

[3H] L-glutamic acid na-ejikọta na tubes microfuge na iko ka a nyochara na nchekwa anọ.Njikọ ndabere na ihe ndị a bụ ihe a na-apụghị ileghara anya, mana abawanyela site na centrifugation ma ọ bụ suction na Tris-HCl na Tris-citrate buffer.Njikọ a pere mpe ma ọ bụ Mgbe HEPES-KOH, ma ọ bụ ihe nchekwa Tris-acetate ka ejiri mee ihe.[3H] L-glutamate na-ejikọta na tubes microfuge bụ L-ma ọ bụghị D-isomers nke glutamate na aspartate.DL-2-amino-7-phosphonoheptanoic acid egbochighị njikọ ahụ.Ogige ndị ọzọ gosipụtara mgbochi dị ala na nke na-agafeghị oke bụ: N-methyl-D-aspartate, qusqualate, L-glutamic acid diethyl ester, N-methyl-L-aspartate, kainate, na 2-amino-4-phosphonobutyrate.Mgbochi ụbụrụ nke oke emebighị emebi gbochiri njide.E nwetara njikọ nke glutamate dabere na protein [3H] site na nkwadebe akpụkpọ ahụ jụrụ oyi ugboro ugboro mgbe ejikọtara ya na nchekwa Tris-acetate.A na-atụ aro na a ga-eji Tris-acetate ma ọ bụ HEPES-KOH buffer mee ihe na glutamate bin ding assay.Ọ bụrụ na ejiri Tris-HCl ma ọ bụ Tris-citrate buffer, a ga-eme nnwale njikwa kwesịrị ekwesị iji dozie maka ijikọ na tubes microfuge ma ọ bụ ihe nzacha eriri iko.