ʻenehana peptide FRET

ʻO ka hoʻololi ʻana i ka ikehu ʻo Fluorescence resonance (FRET)

ʻO ka Fluorescence Resonance Energy Transfer (FRET) kahi kaʻina hana hoʻololi ikehu ʻole i hoʻololi ʻia ka ikehu mokuʻāina i ka ʻae ʻana ma o ka launa pū ʻana o nā kāne uila intermolecular.ʻAʻole pili kēia kaʻina hana i nā kiʻi kiʻi a no laila ʻaʻole ia-radiative.Loaʻa i kēia assay nā pōmaikaʻi o ka wikiwiki, maʻalahi a maʻalahi.

Hiki ke like ke kala i ho'ohana 'ia ma ka FRET assay.Akā i ka nui o nā noi, hoʻohana maoli ʻia nā kala like ʻole.I ka pōkole, ʻo ka hoʻololi ʻana o ka ikehu resonance luminous ka hoʻoili ʻana o nā dipoles ʻelua mai ka mea hāʻawi (dye 1) i ka mea ʻae (dye 2) ke hauʻoli ka hui hāʻawi.Ma keʻano holoʻokoʻa, ʻoi aku ka laulā o ka hui Donor fluorophore me ka spectrum absorption o ka hui Acceptor."Inā kūpono ka mamao ma waena o nā fluorophores ʻelua (10 - 100 A), hiki ke ʻike ʻia ka hoʻololi ʻana o ka ikehu fluorophore mai ka mea hāʻawi aku i ka mea ʻae."ʻO ke ʻano o ka hoʻoili ʻana o ka ikehu e pili ana i ke ʻano kemika o ka mea loaʻa:

1. Ua hoʻololi ʻia i molecular vibration, ʻo ia hoʻi, nalo ka mālamalama kukui o ka hoʻoili ikehu.(ʻO ka mea loaʻa he mea hoʻopau māmā)

2. ʻOi aku ka ikaika o ka hoʻokuʻu ʻana ma mua o ka mea loaʻa iā ia iho, e hopena i ka hoʻololi ʻulaʻula i ka spectrum fluorescence lua.(He mau mea hoʻopuka luminous nā mea hoʻokipa).

ʻO ka hui hāʻawi (EDANS) a me ka ʻae ʻae (DABCYL) i hui like ʻia me ka substrate kūlohelohe o ka HIV protease, a ke hemo ʻole ka substrate, hiki i ka DABCYL ke kinai i ka EDANS a laila hiki ke ʻike ʻole ʻia i ka fluorine.Ma ka hemo ʻana o ka protease HIV-1, ʻaʻole hoʻopau ʻia ʻo EDANS e DABCYL, a hiki ke ʻike ʻia nā luciferases EDANS.Hiki ke nānā ʻia ka loaʻa ʻana o ka protease inhibitors e nā loli i ka ikaika fluorescence o EDANS.

ʻO nā peptides FRET nā mea hana maʻalahi e aʻo i ka peptidase nonspecificity.No ka mea hiki ke nānā mau ʻia kāna kaʻina hana, hāʻawi ia i kahi ala kūpono no ka ʻike ʻana i ka hana enzyme.Hāʻawi ka ʻōlinolino i hana ʻia ma hope o ka hydrolysis o nā paʻa peptide e ka mea hāʻawi / ʻae i kahi ana o ka hana enzyme ma nā ʻano nanomolar.Ke paʻa ka peptide FRET, hōʻike ia i ka nalowale koke ʻana o ka uila i loko, akā i ka wā e haki ai kekahi paʻa peptide e kū pono ana i ka mea hāʻawi / ʻae, hoʻokuʻu ia i kahi uila, hiki ke ʻike mau ʻia a hiki ke helu ʻia ka hana enzyme.