Ukudluliselwa kwamandla eFluorescence resonance (FRET)
I-Fluorescence resonance energy transfer (FRET) yinkqubo yokudlulisa amandla e-non-radiative apho umnikeli wamandla omoya ochulumancisayo adluliselwa kumamkeli wombuso ochulumancisayo ngokusebenzisana kwezibini zombane ze-intermolecular.Le nkqubo ayibandakanyi iifotoni kwaye ngoko ayinayo i-radiative.Olu vavanyo lunoncedo lokukhawuleza, uvakalelo kunye nokulula.
Idayi esetyenziswe kwi-FRET assay inokufana.Kodwa kwizicelo ezininzi, iidayi ezahlukeneyo zisetyenziswa ngokwenene.Ngamafutshane, ukuhanjiswa kwamandla okukhanya okukhanyayo kukudluliselwa kwesibini se-dipoles ukusuka kumnikeli (udayi 1) kumamkeli (udayi 2) xa iqela labaxhasi livuya.Ngokubanzi, i-spectrum ephumayo yeqela le-Donor fluorophore liwelana kunye ne-spectrum yokufunxa yeqela le-Acceptor."Xa umgama phakathi kwee-fluorophores ezimbini ufanelekile (10 - 100 A), ukuhanjiswa kwamandla e-fluorophore ukusuka kumnikezeli ukuya kumamkeli kunokubonwa."Indlela yokudluliselwa kwamandla ixhomekeke kwisakhiwo sekhemikhali ye-receptor:
1. Iguqulwa ibe yi-vibration ye-molecular, oko kukuthi, ukukhanya okukhanyayo kokuhanjiswa kwamandla kuyanyamalala.(I-receptor yinto yokucima ukukhanya)
2. Ukukhutshwa kunamandla ngakumbi kune-receptor ngokwayo, okukhokelela kwi-redshift kwi-spectrum ye-fluorescence spectrum.(Ii-receptors zi-emitters ezikhanyayo).
Iqela labanikeli (EDANS) kunye nejini yokwamkela (DABCYL) zidityaniswe ngokufanayo kwi-substrate yendalo ye-HIV protease, kwaye xa i-substrate ingaqhawulwa, i-DABCYL inokucima i-EDANS kwaye emva koko ingabonakali kwi-fluorine.Ekuqhawulweni kwe-HIV-1 protease, i-EDANS ayisacinywa yi-DABCYL, kwaye i-EDANS luciferases inokufunyaniswa emva koko.Ukufumaneka kwe-protease inhibitors kunokujongwa ngotshintsho kwi-fluorescence intensity ye-EDANS.
I-FRET peptides zizixhobo ezifanelekileyo zokufunda i-peptidase engacacanga.Kuba inkqubo yokusabela kwayo inokujongwa ngokuqhubekayo, ibonelela ngendlela efanelekileyo yokubona umsebenzi we-enzyme.I-sheen eveliswe emva kwe-hydrolysis ye-peptide bonds ngumnikeli / owamkelayo inika umlinganiselo womsebenzi we-enzyme kwi-nanomolar concentrations.Xa i-peptide ye-FRET ingabonakali, ibonisa ukunyamalala ngokukhawuleza kwe-flash yangaphakathi, kodwa xa nayiphi na ibhondi ye-peptide echasene nomnikezeli / i-acceptor iphuka, ikhupha i-flash, enokuthi ibonwe ngokuqhubekayo kwaye umsebenzi we-enzyme unokulinganiswa ngoko.
Ixesha lokuposa: Aug-14-2023