Chromotek/F2H®Kit Androgen Receptor苏州蚂蚁淘生物科技有限公司

Chromotek/F2H-KitAndrogenReceptorAnalyzemodulatorsofthehumanAndrogenReceptorDetectearlystepsofAndrogenReceptoractivation

Chromotek/F2H-KitAndrogenReceptor

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Chromotek/F2H-KitAndrogenReceptor

AnalyzemodulatorsofthehumanAndrogenReceptor

DetectearlystepsofAndrogenReceptoractivation

Visualizedinlivemammaliancells

Real-timeagonistsandantagonistsscreening

Convenientreadoutusinganepi-fluorescencemicroscope

ThehumanAndrogenreceptor(AR)isanimportanttargetfordevelopingdrugtherapiescombatingprostatecancer.TheveryearlyeventinARactivation,namelytheintramolecularinteractionbetweentheN-andC-terminusofAR,cannowbeanalyzedwiththeChromoTekFluorescent-TwoHybrid(F2H)AR:wild-type(wt),aswellasseveralmutantARformscanbeinvestigated.

Chromotek/F2H-KitAndrogenReceptor

Assayprinciple:

ARisexpressedastwoseparatedomains:AR-LBD(ligandbindingdomain),comprisingtheARsC-terminus,eitherwtormut,fusedtoGFP:AR-LBD-GFP

AR-NTD(N-terminaldomain),comprisingtheARsN-terminus,fusedtoRFP:AR-NTD-RFP

F2H-BHKcellsexpresscomponentsoftheprotein-proteininteractionplatform,whichrecruitsGFP-taggedAR-LBDtoaspecificlocationinthenucleus

AR-LBD-GFPandAR-NTD-RFPconstructsareco-transfectedintotheF2H-BHKcells

AR-LBD-GFPformsabrightgreenspotatthespecificlocationinthenucleiwhereasAR-NTD-RFPisdiffuselydistributedinthenucleus

Uponstimulationbyanagonist(e.g.DHT),immobilizedAR-LBD-GFPundergoesaconformationchangeandrecruitsAR-NTD-RFP:AR-NTD-RFPformsabrightredspotsuperimposedwiththegreenspot.

Results:

Greenspotispresent,redspotisabsent=assayworks:nointeraction

Greenspotispresent,redspotispresentatthesamelocation=assayworks:interaction

Figure:AR-LBD-GFP(topleft)formsbrightgreenspotsatthegeneticPlatforminnucleiofF2H-BHKcells.BeforeadditionofDHT,AR-NTD-RFP(bottomleft)isdispersedinthenuclei.Upon20minincubationwith10nMDHT,theredspotsappear(bottomright,arrows)atthelocationofthegreenspots,indicatinginductionoftheinteractionbetweenAR-LBDandAR-NTD.

ReversibleScreening:

TheF2HARassayisfullyreversible:theARwtinteractioncanbeinducedbyanagonist(e.g.0.25nMDHT)andthensubsequentlydisruptedbyadditionofapotentantagonist(e.g.10Mbicalutamide).