Fluorescence resonance energy transfer (FRET)
Fluorescence resonance energy transfer (FRET) inzira isiri-radiative yekutamisa simba umo mupi akafara simba renyika inotamirwa kune anogamuchira inonakidzwa nyika kuburikidza nekudyidzana kwe intermolecular magetsi vakaroorana.Iyi nzira haisanganisi mafotoni uye saka haisi-radiative.Iyi assay ine zvakanakira kuve nekukurumidza, kunzwira uye nyore.
Dhayi inoshandiswa muFRET assay inogona kufanana.Asi mumashandisirwo mazhinji, madhayi akasiyana anoshandiswa chaizvo.Muchidimbu, kuendeswa kwesimba rinopenya resonance ndiko kuendeswa kwepeya ye dipoles kubva kune mupi (dhayi 1) kune anogamuchira (dhayi 2) apo boka revanopa rinenge richifara.Kazhinji, iyo emission spectrum yeDonor fluorophore boka inopindirana neiyo absorption spectrum yeAcceptor boka."Kana chinhambwe chiri pakati pemafluorophore maviri chakakodzera (10 - 100 A), kutamiswa kwesimba refluorophore kubva kumupi kuenda kune anogashira kunogona kucherechedzwa."Iyo nzira yekufambisa simba inotsamira pane kemikari chimiro cheiyo receptor:
1. Inoshandurwa kuita molecular vibration, kureva kuti, chiedza chinopenya chekufambisa simba chinopera.(Iyo receptor inodzima mwenje)
2. Kubuda kwacho kwakanyanyisa kudarika receptor pachayo, zvichiita kuti pave ne redshift musecondary fluorescence spectrum.”(Receptors ndiwo emitter inopenya).
Boka revanopa (EDANS) nejena rekugamuchira (DABCYL) zvakabatanidzwa zvakafanana kune yakasikwa substrate yeHIV protease, uye kana iyo substrate isina kubviswa, DABCYL inogona kudzima EDANS uye yobva yatadza kuoneka kune fluorine.Pakubviswa kweHIV-1 protease, EDANS haichadzimwa neDABCYL, uye EDANS luciferases inogona kuonekwa.Kuwanikwa kweprotease inhibitors kunogona kuongororwa nekuchinja kwe fluorescence intensity yeEDANS.
FRET peptides maturusi ari nyore kudzidza peptidase nonspecificity.Sezvo maitiro ayo ekuita achigona kuramba achitariswa, inopa nzira iri nyore yekuona chiitiko che enzyme.Iyo sheen inogadzirwa mushure meiyo hydrolysis ye peptide zvisungo neanopa / anogamuchira inopa chiyero chebasa re enzyme pane nanomolar concentrations.Kana iyo FRET peptide yakamira, inoratidza kunyangarika kamwe kamwe kwemukati flash, asi kana chero peptide bond yakatarisana nemupi / mugamuchiri ikaputsika, inoburitsa flash, iyo inogona kuonekwa nguva dzose uye basa re enzyme rinogona kuverengerwa.
Nguva yekutumira: Aug-14-2023