Nā huahana

Hoʻohana pinepine ʻia ka paʻakai Tris acetate i ka hoʻomākaukau ʻana i ka buffer TAE (Tris-acetate-EDTA), i hoʻohana ʻia ma ke ʻano he paʻakai holo a i nā gels agarose.Hoʻohana ʻia ʻo Tris Acetate-EDTA buffer no ka DNA agarose gel electrophoresis akā hoʻohana ʻia no ka non-denaturing RNA agarose gel electrophoresis.ʻOi aku ka wikiwiki o ka DNA stranded ʻelua i ka TAE ma mua o nā pale ʻē aʻe a hiki ke luhi i ka wā electrophoresis lōʻihi.Hiki ke hoʻoponopono i ka hiki ke hoʻopaʻa haʻahaʻa i ka holo ʻana a i ʻole ka hoʻololi ʻana i ka pale i ka wā electrophoresis lōʻihi.Hiki ke hoʻohana ʻia ma nā ʻano ʻokoʻa like ʻole e aʻo i ka neʻe ʻana o ka DNA i ka hopena.Ma muli o ka borate i loko o ka TBE buffer (Tris-Borate-EDTA Buffer, 10X Powder Pack, sc-296651) he mea paʻa ikaika no nā enzymes he nui, ua ʻōlelo ʻia ʻo TAE buffer i ka nānā ʻana i nā noi enzymatic no ka laʻana DNA.ʻO ka paʻakai Tris acetate kekahi mea pale me ka naʻau kiʻekiʻe i ka ATP assays me firefly luciferase.

Hoʻohana: ʻO TAE holo ʻana ka mea hoʻohana maʻamau no ka DNA agarose gel electrophoresis, a hoʻohana ʻia no ka RNA agarose gel electrophoresis.ʻOi aku ka wikiwiki o ka DNA stranded ʻelua ma TAE ma mua o nā pale ʻē aʻe, akā ʻaʻole hiki ke ʻauʻau ma muli o ka pau ʻana o nā ion buffer i ka wā electrophoresis lōʻihi.Hiki ke hoʻopaʻi ʻia ke kaʻa hoʻopaʻa haʻahaʻa i ke kaʻa uila a i ʻole ka hoʻololi ʻana i ka wā electrophoresis lōʻihi.2 E hoʻoheheʻe i ka pale TAE i hoʻopaʻa ʻia no ka loaʻa ʻana o 1 mMTAE paʻa me 40 mM Tris acetate a me 1 mM EDTA, pH 8.3.Hiki ke hoʻohana ʻia ka buffer 1 mMTAE i nā gels agarose a ma ke ʻano he buffer holo.No ka hoʻonā kiʻekiʻe, ʻoi aku ka haʻahaʻa o ka volta i hoʻopili ʻia ma mua o 5V/cm (ka mamao ma waena o nā electrodes unit).

Noi: ʻO ka TAE holo ʻana ka mea hoʻohana maʻamau no ka DNA agarose gel electrophoresis i Chemicalbook gel, a hoʻohana ʻia no ka non-denaturing RNA agarose gel electrophoresis.ʻOi aku ka wikiwiki o ka DNA stranded ʻelua ma TAE ma mua o nā pale ʻē aʻe, akā ʻaʻole hiki ke ʻauʻau ma muli o ka pau ʻana o nā ion buffer i ka wā electrophoresis lōʻihi.Hiki ke hoʻopaʻi ʻia ke kaʻa hoʻopaʻa haʻahaʻa i ke kaʻa uila a i ʻole ka hoʻololi ʻana i ka wā electrophoresis lōʻihi.Ua hoʻoheheʻe ʻia ka pahu TAE i hoʻopaʻa ʻia no ka loaʻa ʻana o 1 mMTAE buffer i loaʻa iā 40 mM Tris acetate a me 1 mM EDTA, pH 8.3.Hiki ke hoʻohana ʻia ka buffer 1 mMTAE i nā gels agarose a ma ke ʻano he buffer holo.No ka hoʻonā kiʻekiʻe, ʻoi aku ka haʻahaʻa o ka volta i hoʻopili ʻia ma mua o 5V/cm (ka mamao ma waena o nā electrodes unit).

Hoʻohana: I ka ʻike ʻana o ATP me firefly luciferase, ʻo kēia huahana ka mea paʻa paʻa loa;ʻike paʻa glutamate.

ʻO ka hoʻopaʻa ʻana o [3H]l-glutamic acid i nā mea hoʻokipa ʻole.

[3H] L-glutamic acid i hoʻopaʻa ʻia i nā paipu microfuge a me ke aniani i noiʻi ʻia i loko o ʻehā mau pale.ʻAʻole hiki ke hoʻopaʻa ʻia i ka hope i kēia mau mea, akā ua hoʻonui ʻia e ka centrifugation a i ʻole suction ma Tris-HCl a me Tris-citrate buffer.ʻOi aku ka liʻiliʻi o kēia paʻa ʻana a i ʻole I ka wā i hoʻohana ʻia ai ka HEPES-KOH, a i ʻole Tris-acetate buffer.[3H] L-glutamate i hoʻopaʻa ʻia i nā paipu microfuge i kāohi ʻia e L- akā ʻaʻole D-isomers o ka glutamate a me ka aspartate.ʻAʻole i kāohi ka DL-2-amino-7-phosphonoheptanoic acid i ka hoʻopaʻa ʻana.ʻO nā pūhui ʻē aʻe i hōʻike i ka haʻahaʻa haʻahaʻa a me ka haʻahaʻa haʻahaʻa: N-methyl-D-aspartate, quisqualate, L-glutamic acid diethyl ester, N-methyl-L-aspartate, kainate, a me 2-amino-4 -phosphonobutyrate.Kāohi ʻia ka paʻa ʻana e nā membrane lolo ʻiole denatured.Ua loaʻa kahi hoʻopaʻa paʻa [3H] glutamate e pili ana i ka protein me ka hoʻomākaukau ʻana i ka membrane i hoʻoheheʻe pinepine ʻia i ka wā i hana ʻia ai ka hoʻopaʻa ʻana ma Tris-acetate buffer.Manaʻo ʻia e hoʻohana ʻia ʻo Tris-acetate a i ʻole HEPES -KOH buffer i ka glutamate bin ding assay.Inā hoʻohana ʻia ʻo Tris-HCl a i ʻole Tris-citrate buffer, pono e hana ʻia ka hoʻokolohua hoʻomalu kūpono e hoʻoponopono ai no ka hoʻopaʻa ʻana i nā paipu microfuge a i ʻole nā ​​kānana fiber aniani.