1.Runsamplesoutonagel.Forbacteriallyexpressedproteins,generally5uLisplenty(1mlcellculture;cellsresUSPededin50ulloADIngbuffer).Runthegels(BioRadminigels)at195Vforapproximately40min.(untilsamplesrunclosetothebottomofthegel).

2.Transfertheproteinsfromthegelontonitrocellulose.FortheBioRadsetup,thecaseshouldbesetupasfollows:blacksidedown,then3MScotchBritePad,thenblottingpaper,gel,nitrocellulose,2ndblottingpaper,2ndScotchBritePad,andtheclearsideofthecase.Putthecaseintheholder,blacksideofthecasefacingblacksideoftheholder.Runthetransferat100Vfor1hour.

3.Meanwhile,prepare500mLofACBuffer(+Tween):

50mLglycerol(=10%glycerol)

10mL5MNaCl(=100mMNaCl)

10mL1MTris,pH7.6(=20mMTris)

1mL0.5MEDTA(=0.5mMEDTA)

5mL10%Tween-20(=0.1%Tween-20)

putonice

b)Make50mL(ormore)of2%milkpowdersolution:

50mLACBuffer

1gmilkpowder

-putonarockertodissolvethemilkpowder(maytake20-30minutes).Thenputonice

4.Maketheprobe,usingtheTnT(Promega)ReticulocyteLysatekit.

Setupeithera25uLreactionora50uLreaction,dependingonthesizeoftrayyou"llbeusingforwashesandprobing.Belowistherecipefora50uLreactionmix:

25uLReticulocytelysate(Iusealittlemore,~27uL)

2uLReactionBuffer

1uLT7(orT3)polymerase(orotherpolymerase)

1uLaminoacidsminusmethionine(ormissingotheraminoacid)

1uLRNAGuard

4uL35S-met(orotherlabelledaminoacid)

16uLDNA+ddH2O(1-2ugDNA)

Spindownthesample(pulsespin)toremoveairbubbles.Letthereactionproceedatroomtemperaturefor1hour,10minutes(canbelongerorshorter,dependingontheprotein).

Block,Probe,andWash

5.Afterthetransferiscomplete,puttheblotintoatray.Keepthesidethatwastouchingthegelup).Do1-2quickwasheswith1XPBStoremovetheSDS.Then,doaquickrinseinACBuffer,toremovethePBS.Pouronthe2%milkpowder(justenoughtocovertheblot),putalidonthedish,androcktheblotat4Cfor1hour.Thisistheblockingstep.

6.Meanwhile,setup2spincolumns:

Use1ccsyringes,removethecapandtheplunger.Putinglasswooltofilltheopeningatthebottom,pushdownwiththeplunger.Putthesyringeina15mLfalcontube.AddBioRadG25resin,storedat4CinTEbuffer,tothesyringe,fillingtothetop(avoidairbubbles!).Spindownat2000rpmfor3minutes.ThenrefillwithG-25,andrepeatthespincycle.(Atthispoint,theG-25shouldbepackeddownsuchthatitoccupiesabout0.8ml.

Addtothecolumns100uLACBuffer,spinat2000rpmfor3min.Repeattwomoretimes.

7.Whentheprobelabelingreactionisfinished,dilutethesampleusingACBuffertomakeafinalvolumeof100uL.Loadthisonthespincolumnandspinat2000rpmfor3minutesinafreshfalcontube.Collecttheflow-through.Thisprocessremovesunincorporatednucleotides.

8.Whenthereareapproximately20minutesleftintheblockingstep,itistimetopreparetheprobemix.Atthispoint,youmaywanttocollecta2uLsampleofyourprobe(fromthe100ul),andcombineitwith10uL1XSDSgelloadingbuffer.Youcanthenrunthissampleonagel,andputfilmonthedriedgeltoseeiftheprobelabellingstepactuallyworked.

Thevolumeoftheprobemixdependsonyourinitialprobereactionmixvolume,andthesizeoftraythatyouareusing.Forexample,ifyousetupa50uLinitialreaction,youcanuseupto20mLofprobemix.Fora25uLreaction,usenomorethan10mlofprobemix.

Lettheprobeincubateinthemixfortheremaining20minutes,onice.

9.Whentheblockingstepisover,pouroffthemilkpowdersolution,andpourontheprobemix.Coverthetray.Rocktheblotinthecoldroomfor2-3hours.

10.Whenyouarefinishedprobing,pourtheprobemixintoasuitableradioactivewastecontainer.Thendo2quickwasheswithACBuffer.Washwithremaining2%milkpowdersolution(+DTT),andlettheblotrockinthecoldroomfor15min.(makesureyouhaveenoughsolutiontocovertheblotcompletely).

11.DoaquickwashinACBuffer,andfollowwith4x20minwashesinACbuffer,inthecoldroom.(useatleastenoughtocovertheblot)

12.Lettheblotdryonblottingpaper.After30-40minutes,transfertoanewpieceofblottingpaper.Addbabypowdertopreventsticking.SpreadGENTLY.Makesurethesideofthemembranethatwasincontactwiththegelisthesidethatisup.Putonx-rayfilm,andexpose.

note:

-ifpreferred,theblockingstepcangoovernight(thestepbeforeprobing),ratherthanforanhour.So,ifyouarepressedfortime,thisisonepointwhereyoucanspreadtheFarWesternovertwodays.

-Denaturation/Renaturationstepscanalsobetriedtooptomizesignal:noise.Forexample,rightafterthetransfer,theblotiswashedwith6MGuanidine-HClinACBufferplus2%milkpowder(andDTT)for30minutesatroomtemperature(use8Mstock).Thenitiswashedin3MGuanidine-HClin2%milkpowder(inACBuffer)for30minutesatRT;then1MGuanidine-HClin2%milkpowder(inACBuffer)for30minutesat4C,andthenin0.1Mfor30minutesat4C.Finally,itisputin2%milkpowdersolution,inACBuffer,withDTT,overnightat4C.Proceedwithprobingthenextmorning.

BelowisatableshowinghowtomaketheGuanidine-HClsolutions.

FarWesternblotofFTZpolypeptidesexpressedinbacteriaanddetectedby35SFTZ-F1