SDSpolyacrylamidegelelectrophoresis(SDSPAGE)willbeusedtoassessthepurificationprocessandtodeterminetheapparentmolecularweightsofthethreeapoproteins.

SDS-Polyacrylamidegelelectrophoresis

Electrophoresisistheprocessinwhichchargedparticlesmigratethroughasolidorliquidmatrixinresponsetoapplicationofanelectricfield.Rateofparticlemovementisproportionaltothecharge:massratiooftheparticleandtoitsfrictionalresistance.Largerparticlesmovemoreslowly,andhighlychargedparticlesmovemorequickly.Inproteinelectrophoresis,thesefactorstendtobalanceout.Sizeandchargeofaproteindetermineitselectrophoreticmobility.Ifproteinsareseparatedthroughagelmatrixwithvaryingporesize,migrationdependsonthesizeandshapeoftheprotein.Smallerproteinsareretainedless,andthusmovefaster.Ontheotherhand,thenetchargeofaproteindependsonthepH.Innativegelelectrophoresis,bothchargeandsizedeterminethemigrationpattern;inthistechniqueexcellentseparationcanbeachieved,butunambiguousinformationabouttheproteinsizecannotbeobtained.

However,proteinswhichhaveasimilarnetchargeseparatenicelyaccordingtotheirsize,providedtheyareofsimilarshape.Forexample,globularproteinsareretainedlessthanrod-likeproteinsofthesamemolecularweight.Experimentally,wecaneliminatetheinfluenceofchargeonproteinmigrationbyprovidingallproteinswithextremenegativecharges.Sodiumdodecylsulphate(SDS),isananionicdetergentthatbindstoproteins.Itsactionistodenaturetheproteinbysolubilizingitandeffectively"coating"itwithanegativecharge.Theeffectbeforeelectrophoresisistoblockproteininteractionswithotherproteins,polymerssuchasnucleicacidsandlipid,todissociatemultimericproteins;andtoalterfoldinginproteinmonomers.SDSnotonlyprovidesproteinswithastrongnegativecharge,butitalsodenaturestheprotein,thusgivingeachproteinaroughlyglobularshape;differencesinproteinshapethatwouldaffectelectrophoreticmobilityareeliminated.

Thegelmatrixmostcommonlyusedforproteinsseparationispolyacrylamide.PolyacrylamidegelsareformedwhenmonomericacrylamideispolymerizedbytheactionofarADIcalformingagent,ammoniumperoxidisulfate(ammoniumpersulfate)andN,N,N",N"-Tetramethylenediamine(TEMED).Thegelformsoptimallyintheabsenceoffreeoxygen,sinceoxygenisastabledi-radicalwhichcanterminatetheradicalinducedpolymerizationreaction.Sinceacrylamidepolymerizestolonglinearproducts,across-linkerisrequiredtoformathree-dimensionalgel.Bisacrylamideservesthisfunction.Poresizewithinthegelisdeterminedbyboth,thetotalacrylamideconcentration(%T;=gacrylamide+gbisacrylamideper100ml)andtherelativeconcentrationofthecross-linkerbisacrylamide(%C;=gbisacrylamideper100g(acrylamide+bisacrylamide).WhileporesizedecreaseswithincreasingT,smallandlargeC-valuesyieldlargepores;thesmallestporesareformedinthepresenceofapprox.5%C,asseeninthiselectronmicrograph.

Gelscanbecastascolumnsorslabs.Foranalyticalpurposes,slabsaremuchmorewidelyused,sincetheyallowtheseparationandcomparisonofmultiplesamples.Ineithercase,a"stacking"gelisplacedonthetopoftheseparating("running")geltosharpenthebandsbeforetheyenterthegel.TheelectrophoresisbufferandthebufferintheseparatinggelhaveahighpH(8.9)andcontainglycine.Incontrast,thestackinggelbufferhasalowpH(6.8)andcontainsCl-.AtthelowpHofthestackinggel,theCl-inthestackinggelarenegativelychargedandhencemovetowardstheanode(+),buttheglycineenteringfromthegelbufferhasonlyaverysmallnegativecharge(pIofglycine~6).Thus,Cl-movesfasterthanglycinate,andwithinthestackinggelazoneoflowanionconcentration(=lowconductivity)forms.Thisleadstoahigherelectricfield,whichacceleratestheproteinssothattheyentertheseparatinggelasanarrowbandattheboundarybetweentheleadingCl-andthetrailingglycinateions.Whentheproteincomplexesreachtherunninggel(pH9.8),theglycinebecomescompletelydissociated,andmigratesatthesamespeedasCl-.Youcanlookataschematicillustrationofthismechanism.

SetupSDS-minigelaccordingtotheBioRadinstructions,asoutlinedbelow.

AssemblingtheGlassPlateSandwiches1.Assemblethegelsandwichonacleansurface.Laythelongerrectangularglassplatedownfirst,thenplacetwospacersofequalthicknessalongtheshortedgesoftherect-angularplate.Next,placetheshorterglassplateontopofthespacerssothatthebottomendsofthespacersandglassplatesarealigned(Figure).Atthispoint,thespacersshouldbestickingupabout5mmabovethelongglassplate.

2.Loosenthefourscrewsontheclampassemblyandstanditupsothatthescrewsarefacingawayfromyou.Firmlygrasptheglassplatesandwichwiththelongerplatefacingawayfromyou,andgentlyslideitintotheclampassemblyalongthefrontfaceoftheacrylicpressureplate.Thelongerglassplateshouldbeagainsttheacrylicpressureplateoftheclampassembly.Tightenthetoptwoscrewsoftheclampassembly.

3.Placetheclampassemblyintothealignmentslotsothattheclampscrewsfaceawayfromyou.Loosenthetoptwoscrewstoallowtheplatesandspacerstosettleagainstthecastingstandbase.InserttheMini-PROTEANIIalignmentcardbetweentheglassplates,inordertopositionthespacersproperly.Gentlytightenbothpairsofscrews.

4.Removethealignmentcard.Pullthecompletedsandwichfromthealignmentslot.Checkthattheplatesandspacersareflushatthebottom.Ifnot,realignthesandwichasinsteps1-3.

5.Usingthelevelingbubble,levelthecastingstandwiththealignmentslotfacingyou.Checktoseethattheremovablegraysiliconegasketsarecleanandfreeofresidualacry-lamidetoinsureagoodseal.Placethesiliconerubbergasketsontopoftheredfoampadsofthecastingstandslots.

6.Transfertheclampassemblytooneofthecastingslotsinthecastingstand.Iftwogelsaretobecast,placetheclampassemblyonthesideoppositethealignmentslottomakealigningthenextsandwicheasier.

7.Attachthesandwichinthefollowingway:Butttheacrylicpressureplateagainstthewallofthecastingslotatthebottom,sotheglassplatesrestontherubbergasket.Snaptheacrylicplateunderneaththeoverhangofthecastingslotbypushingwiththewhiteportionsoftheclamps(seeFigure).Donotpushagainsttheglassplatesorspacers.Thiscouldbreaktheplate.

Note:Itisespeciallyimportanttoassurethattherubbergasketisplacedcorrectly(withnotchfacingglassplate),andthatthebottomisalignedexactlytogiveasmoothseal.Itisrecommendedtofilltheassembledcassettefirstpartiallywithwater,markingthemeniscuswithafelttippen.Ifnoleakageisdetectedin5minutes,pouroutthewaterandremovetheresidualwaterbyinsertingafilterpaper.Youcanthenbegintopourthegel.

Preparationofseparatinggel

Toprepare20mlofhomogeneousgel(thisamountisfortwominigels)withtheconcentrationgivenbelow,Pipetteouttheamountsshown(withtheexceptionofTEMEDandtheSDSsolution)inthefollowingtableintoa250mlsidearmedErlenmeyerflask.Note:DegasthemixturebeforeaddingTEMEDandSDS.

1.Useaplastic10mlpipettetopourthegelintoplates.Pourresolvinggelupto~2cmfromtop.

2.Toavoidexposuretoair,carefullylayerwaterontopoftheresolvinggel.Leavethegeltopolymerize.Asharplinebetweenwaterlayerandgelindicatescompletionofpolymerization.Whilewaitingforthegeltopolymerizeyoucanstartpreparingthestackinggel.

Preparationofstackinggel

Preparea7.5mlof3%stackinggelinasmallbeakerusingthefollowingamountsofappropriatereagents.

3.Whenthepolymerizationofresolvinggeliscomplete,decantthelayerofwater.DryexcesswaterusingKim-wipes.

4.Pourthestackinggelusingapasturepipette.Insertthecombgently.Leavetopolymerizeuntilgelturnsmilky(atleast30min.).

AssemblingtheUpperBufferChamber

Note:Toinsurealeakproofseal,makesurethegrayU-shapedinnercoolingcoregasketsareclean.Inspectthegasketforsmallcutsthatcouldresultinanupperbufferleak.Therearetwosidestothisgasket.Makesurethatthesidewiththenotchisexposedforcontactwiththegelsandwich.

1.Releasetheclampassemblies/gelsandwichesfromthecastingstand.

2.Laytheinnercoolingcoredownflatonalabbench.Withtheglassplatesofthegelsandwichfacingthecoolingcore(andtheclampscrewsfacingout),carefullyslidetheclampassemblywedgesunderneaththelocatorslotsontheinnercoolingcoreuntiltheinnerglassplateofthegelsandwichbuttsupagainstthenotchintheU-shapedgasket(Figure5.1).

Note:LubricatingtheraisedportionsoftheU-shapedgasketwithadropofrunningbufferorwaterhelpstheglassplatesandwichslideinproperly.Whilepushingtheclampassemblyslightlyuptowardthetopofthelocatorslots,snaptheclampassemblyfullyontothecoolingcorebypressingatthebottomoftheclampassemblyuntilthecoolingcorelatchengageseachsideoftheclampassembly.(Donotpulloutoncoolingcorelatchatthesametime.)

Electrophoresis

1.Removethe10dialyzedfractionscollectedafterthedensitygradientcentrifugationandthepooledlipophorinsamplefromthecoldroom.Remove50µleachandplaceintoapre-labeledEppendorftubes.Labelthetubeatthelidwithawaterproofpen;otherwiseyouwon"tbeabletoidentifyyoursamples!

2.Add25µlsamplebuffertoeachfractionandcloselids.TogetherwithonetubeofmolecularweightMarkers,placeinthesampleholderandboilfor2min.Usethefollowingmolecularweights:

3.Inthemeantime,removethecombfromthegel.Markthewellswithafeltpen.Thiswillenableyoutoseethewellsclearlywhentherunningbufferispouredintotheupperchamber.Assembletheupperbufferchamber.

5.Dilute60mlof5xstockofrunningbufferwith240mlofdist.water.Pourcarefullyintotheupperbufferchamberuntilthewellsarecovered.Pourtherestofthebufferintolowerbufferchamberalongthewallsofthecontainer.Makesurenoairbubblesaretrappedunderthegels.Ifpresent,youcanremoveairbubblesusingawirebentatthetip.

6.Havetwostandardmolecularweightmarkers(highandlowrange)ready.Itisnotnecessarytoaddsamplebuffertothemolecularweightmarkerssinceithasalreadybeenadded.Theseshouldbeloadedintothetwocornerwells.

7.UsingacleanHamiltonsyringeloadthesamplesintothewells.Load10µlofeachsample.Forthepooledlipophorinsample,loadthreedifferentamountstoassureagoodbandingpatternfordensitomentricanalysis:e.g.,2µl,5µl,15µl.Rinsethesyringewellwithdist.wateraftereachsample.

8.Placethecoverandattachthepowersupply.Turnonthepowerandsetrunvoltageto125volts.Approximateruntimeisabout1h.

9.whilethethegelisrunning,dropthetubewiththelipophorininliquidnitrogen.

10.PlaceaParafilmontopofthetubeandPiercethefilmwithaneedle.

11.Leaveinalyophilizerjarandattachtothelyophilizer.

12.Freezedry(lyophilize)overnightorlonger.

13.Oncetheelectrophoresisruniscomplete(whenthemarkerdyereachesapproximately1cmfromthebottomofthegel),turnthepoweroff.Removethegelsfromtheupperbufferchamber.Laytheinnercoolingcoreonitssideandremovetheclampassemblybypushingdownonbothsidesofthecoolingcorelatchandupontheclampsuntiltheclampassemblyisreleased.Slidetheclampassemblyawayfromthecoolingcore.Openthescresandremovethegelsandwich.

14.Propopentheglassplate.Removethegelcarefullyandleaveinthestain.Stainovernight.

Day2(youwillneedtocome2or3timesfor10minutes)

1.Decantusedstainintothebottleassigned.Pourdestainandleaveonshakerfor2-3horuntilbandsarevisIBLeinalighterbackground.

2.Thegelscanbestoredindilutedestainsolution(destain:water1:1)inacoveredpetridish,sealedwithparafilm.