REFERENCE:Hoffman,G.,Garrison,T.R.,andDohlman,H.G.,AnalysisofRGSproteinsinSaccharomycescerevisiae,MethodsEnzymol.344:617-631,2002.

1.Growastartercultureat30Cshaking(250rpm)untilitreachessaturation.

***ThecellsshouldbetransformedwithayeastexpressionvectorcontainingthelacZgeneunderthecontroloftheFUS1promoter.Alternately,astraincanbeusedwiththeFUS1-lacZreporterintegratedintothegenome.Thesematerialsareavailablefromanumberofyeastlabs,includingourown.

2.Usingthesaturatedstarterculture,inoculate5to25mloftheappropriatemedia.

***Rarely,atransformedcolonywillnotrespondatalltopheromone.Thecauseisnotknown.Toeliminatethese"flatliner"strainspickseveraldifferentcolonies.Twohoursafterre-inoculationtakeanaliquotofeachandperformasmallscaleassay(plusandminus1doseofalpha-factor).Discardanythatfailtochangecoloraftersubstrateaddition.

3.Growat30Cshaking(250rpm)untiltheOD600nm~0.8(thisisusuallydoneovernight).

***Thisisthetrickiestpartoftheassay,sinceitisdifficulttogetdifferentstrainstoreachOD600nm~0.8atthesametime.Thebestwaytohandlethisistostarta2mlstartercultureabout3-4daysbeforetheassay.Thenightbeforetheassay,starta10to25mlintermediateculture.Themorningoftheassay,measuretheabsorbanceofalloftheintermediateculturesanddilutethemdowntoanOD600nmof0.2inpre-warmedmedia.Thestrainswillnowonlyhavetogothroughtwodoublingsandtheamountofvariancebetweenthemshouldbereduced.IfthestrainsstillreachOD600nm~0.8atdifferenttimes,itisacceptabletoputstrainswhichhavereachedOD600nm~0.8onicewhilewaitingfortheothers.

***Re-checktheabsorbanceofallculturesbeforeproceeding.

4.Aliquot10ulofalpha-factorattheappropriateconcentrationsinto96wellplates.

***Eachstrainshouldbetestedwith8to10differentconcentrationsofalpha-factorintriplicate.

***Agoodsetoffinalalpha-factorconcentrationsfortestingansst2deltamutantstrainis:0,0.00003uM,0.0001uM...0.3uM.ForstrainsexpressingamammalianoryeastRGSprotein,thefinalconcentrationsshouldbe100-300foldhigher.Theconcentrationofalpha-factoraddedtoeachwellmustbe10-foldhigherthanthedesiredfinalconcentration.

***Keepafrozenstockofalpha-factorandmakeanewsetofdilutionseachtimetheassayisperformed,sincealpha-factorissubjecttodegradationuponrepeatedfreeze-thawcyclesorexposuretoroomtemperature.

***Amulti-channelpipettorandaplasticpipettorbasin(FisherScientific,#13681100)ishelpfulforaliquotingsolutions.

5.Add90ulofcellstoeachwell.

6.Incubate90minat30C,shakinggently.

7.Duringtheincubation,preparetheFDGsolution.

Solution#1:1mMFDGstockdilutedin25mMPIPES(pH7.2)

Solution#2:5%TritonX-100dilutedin250mMPIPES(pH7.2)

***MixSolution#1andSolution#2inequalamountsjustpriortouse,andpourintoacleanpipettorbasin.

FDGStock:10mMFDG(Fluoresceindi-b-D-galactopyranoside,Molecularprobes,#F-1179)indimethylsulfoxide(DMSO)(FDGshouldbestoredinDMSOat-20C;itismorestableinsolution.)

8.Afterthe90minincubation,add20ulFDGsolutionperwell.Shakeplatesgentlyandbriefly.

9.Coverinaluminumfoilandincubateat37Cuntilabrightyellowcolorappearsinsomeofthewells.

***Thiscantakefrom10to90min.Donotincubatelongerthan90min.

10.Stopthereactionbyadding20ulof1MNa2CO3perwell.Shaketheplatesgentlyandbriefly.

11.Readtheplateswithafluorescencemulti-wellplatereaderusinganexcitationof485nmandanemissionof530nm.

12.Readtheabsorbanceandnormalizeforcelldensity.

***Alternately,usethefinalabsorbancevalues[obtainedimmediatelybeforealiquotingcellsintothe96wellplate(step3)]tonormalizeforcelldensity.

Updated01/23/02