Bio6–SDS-PAGELab

ObjectivesUponcompletionofthislaboratoryyouwillunderstandhowtoloadandrunproteinsamplesonanSDS-polyacrylamidegel,stainthegel,andanalyzetheresultingbandsofproteinonthegeltoestimatethemolecularweightofeachprotein.

Introduction

SDS-PAGE is a very common laboratory technique used to analyze proteins. The acronym SDS-PAGEstandsforsodiumdodecylsulfate–polyacrylamidegelelectrophoresis.SodiumdodecylsulfateorSDSisadetergentcommonlyusedinbiologylaboratoriestodenatureproteins,i.e.,disruptthe3-dimensionalstructureofproteinswithoutbreakingthepolypeptidebackbone.Polyacrylamideisapolymerpreparedasagelatinousmediumor“gel”throughwhichproteinscanberesolvedbasedonmolecularweight(MW).Electrophoresisreferstothemovementofchargedsolubleparticlessuchasproteinsthroughamediumduringexposuretoanelectricfield.SDS-PAGEisthereforeatechniquebywhichproteinsmovethroughapolyacrylamidegelthatissubjectedtoelectriccurrent.The rate at which a protein moves through the microscopic pores of a polyacrylamide gel duringelectrophoresisisdependentonthreephysicalproperties–molecularweight,3-dimensionalshape,andnetcharge.SeparationofproteinsbasedsolelyonthepropertyofMWispossibleonlyifthevariablesof3-dimensionalshapeandnetchargeareeliminated.ThisisaccomplishedbythedetergentSDS,whichduetoitsamphiphilicpropertiesofbeinghydrophobicononeendandchargedontheotherisabletodisruptallnon-covalentinteractionsinaprotein–i.e.,denaturetheprotein.Thisresultsinunfoldedorlinearpolypeptidesthathaveanetnegativelychargeasshownbelow:

SDS thuseliminatesanydifferences in shapeandoverall chargeamongproteins, leavingonevariablephysicalproperty–molecularweight–toinfluencetherateatwhichaproteinmovesthroughthegel.

57

Asshownbelow,SDS-PAGEisrunverticallyandthegelconsistsofa“stackinggel”ontopofa“resolvinggel”(aka“runninggel”).Bothendsofthegelareincontactwithanelectrolytebuffersolutioncontainingthepositiveandnegativeelectrodeswhichgenerateelectriccurrentthroughthegel.

Thestackinggelcontainscavitiescalledwells intowhichyouwill loadproteinsamples. Whenelectriccurrentisapplied,negativelychargedsolutessuchasSDS-treatedproteinswillexperienceelectromotiveforcetowardtheoppositelychargedpositiveelectrodeandthusmoveintothestackinggel.ThestackinggelhasanacrylamidepercentageandpHthatensuresallproteinsentertheresolvinggelatthesametime.Onceintheresolvinggel,proteinswillmigrateatarateinverselyproportionaltoMW.Whenthegelisfinishedrunning,itisstainedtorevealthepositionofeachproteinonthegelasshownbelow:

+

–

58

Eachstainedbandonthegelcorrespondstoaoneormoretypesofproteinwith thesamemolecularweight(keepinmindthateachbandcontainsbillionsofproteinsofthatMW).Thelargertheprotein,thesloweritmovesthroughthegelandthusthehigheritsposition.Smallerproteinsmoveatfasterrateandareseenasbandsfurtherdownthegel.AproteinMWstandard(acollectionofproteinsofknownsize)isalwaysrunonthegelandusedtoestimatethesizesofproteinsintheotherlanes.

Part1:RunninganSDS-PAGEgelTobeginthis labyouwilldenatureandloadseveraldifferentproteinsamplesonapolyacrylamidegelafterwhichyouwillrunthegelasdescribed.PreparinganacrylamidegelforSDS-PAGEisabittricky,sothepolyacrylamidegelshavebeenpreparedforyou.Todenatureyourproteinsitisessentialthatyouaddsample loadingbuffer and thenboil the sample. Thesample loadingbuffer contains severalkeycomponentsneededtofullydenaturetheproteinsandhavethemrunthroughthegelproperly:

• SDStodenaturethepolypeptides• Dithiothreitol(DTT)tobreakdisulfidebonds(covalentbondsbetweencysteines)• Glyceroltomakethesampledenseenoughtosinkintothewell• BromophenolBlue,anegativelychargeddyetomonitorgelprogress

Theloadingdyeispreparedata2Xconcentrationsothatitcanbedilutedto1Xwhenmixedwiththeproteinsample.Onceloadingdyehasbeenadded,theheatfromboilingfacilitatesdenaturationoftheproteinsandbreakingofdisulfidebonds.Disulfidebonds(refertochapter5ofyourtextbook)aretheonlycovalentinteractionsformedbetweenaminoacidRgroupsandarenotdisruptedbySDS,thustheneedforDTT.Onlyinthisfullydenaturedstatewilleachproteinsamplemovethroughthegelataratedependentonmolecularweightonly.Loadingsamplesintothewellsofthegelcanbechallengingsincethegelisverythin,toothintoinsert the pipet tip directly into the well. Asshowninthepicture,youwillneedtogentlyrestthepipettipontopoftheinnerplateatananglesothatthepipettipisrestingjustabovethewellyouwanttoload.Itisespeciallyimportantthatyoudonotpressthepipettipdownbetweentheplates,otherwiseyoumayprythemapart.Ifthishappens,evenjustalittlebit,yoursampleswillleakdownbetweenthegelandtheplateandbelost.Beforeyouloadyourproteinsamplesyouwillloadapracticesamplesoyouwillhavesomeexperiencebeforeloadingyourrealsamples.

59

Exercise1–LoadingandrunningyourgelAllofthematerialsyouwillneedcanbefoundoncartsatthesideofthelabwhereyouwillobtainanicebucketandatrayofmaterials.Onceyouhaveallyourmaterials,besuretohalffillthebeakerwithhottapwaterandputitonahotplatesettothemaximumsoboilingwaterwillbereadywhenyouneedit:

1. Each protein sample is 5 µl. Add 2X sample loading buffer to each protein sample so the finalconcentrationis1X(calculatethisasyoudidinthe“MetricSystem”lab).

2. Tapeachtubegentlytomixandboilthesamplesalongwiththemolecularweightmarkerfor5minutes(theproteinmolecularweightmarkeris5µlandalreadycontains1Xsampleloadingbuffer).

3. Identifythewellsyouplantouseforyourproteinsamples(avoidusingdeformedwells),andindicate

inyourlabnotebookhowyouwillloadyourgel.Eachpersonshouldpracticeloading10µlof1Xsampleloadingbufferintheunusedlanes.Yourinstructorwilldemonstratehowtodothis.

4. Loadalloftheproteinmolecularweightmarkerandeachproteinintotheirdesignatedlanes.

5. Placethecoveronthegelapparatusandconnecttheredandblackplugstothecorrespondingcolors

inthepowersupply.NOTE:Ifthebufferlevelintheupperchamberisnotabovetheinnerplateofthegel,therewillbenoflowofelectriccurrent.

6. Besurethepowersupplyispluggedinandsetthetoggleswitchbelowthedigitaldisplayto“Volts”,

theRangeSelectswitchto“Low”andtheVoltageSelectknobto“Min.”

7. Turnonthepower(toggleswitchonupperleft)andadjusttheVoltageSelectknobuntilthedigitaldisplayreadsbetween110and120volts.NOTE:Ifthegelisrunningproperlyyouwillseeacurtainoftinybubblesrisingfromthewireinthe

upperbufferchamber.Youshouldalsoseeamuchloweramountof“bubbling”fromthewireinthelowerbufferchamber.Ifyoudon’tseethis,besuretoconsultyourinstructor.

8. Letthegelrununtilthebromophenolbluedyeis~1cmfromthebottomofthegel(~90minutes).9. Whentheruniscomplete,turntheVoltageSelectknowto“Min.”andturnoffthepower.

60

Part2:STAININGPROTEINSINAPOLYACRYLAMIDEGEL

TovisualizetheproteinsinyourpolyacrylamidegelyouwilluseastaincalledCoomassieBrilliantBlue.Thisisnottheonlyproteinstainonecoulduse,howeveritisaverycommonlyusedstaintoviewproteinsonpolyacrylamidegels.Thestainingprocessmayrequireanovernightincubationsoyoumaynotseetheproteinbandsonyourgeluntilthenextlaboratorysession.Exercise2–Stainingapolyacrylamidegel

Besuretoweargloveswhenyouhandlethegelandstainingsolutions,andtocleanupasindicatedbyyourinstructor:

1. WhenthegelruniscompletebesuretheVoltageSelectknobissetto“Min.”andthepowerisoff,thenremovethecoverofthegelapparatus.

2. Carrythegelapparatustothesinkanddiscardtherunningbufferfrombothchambersintothesink.

3. Loosentheclampsholdingthegelinplace,carefullyremovetheplatescontainingyourgelandlayitona

flatsurfacewiththetallerouterglassplatefacingup.

4. Usetheplasticspatulasuppliedasawedgetocarefullypryapartthe2plates.Thegelshouldsticktooneplateortheotherasyoudoso.Ifitseemstosticktoboth,askforassistancefromyourinstructor.

5. Place~100mloffixativesolutioninthestainingtrayprovided.

6. Gentlyseparateabottomcornerofthegelfromtheglassplateusingthespatula,carefullypeelthegelofftheplate(it’sOKifthestackinggeltearsoff,youwon’tneedit)andplaceitinthefixativesolution.Ifthegelcurlsup,carefullyunravelitwithyourglovedhanduntilitflattensout.

7. PlaceaProteinInstaStaincard(whichcontainsCoomassieBrilliantBluestain)facedowninthefixativesolutionoverthegel,placethelidonthecontainer(besuretolabelthelidwithyourgroupnumber)andplacethecontainerontheslowrockeratthesideofthelab.Leavefor1to3hoursorovernight.

8. Whentheproteinbandsareclearlyvisible,photographyourgelasindicatedbyyourinstructorbeingsuretoincludearuleralignedwiththetopoftheresolvinggel.Storeanimageofthegelinyourlabnotebook.

Part3:ESTIMATINGTHEMOLECULARWEIGHTOFPROTEINSONAPOLYACRYLAMIDEGEL

Youwillusetheproteinmolecularweightstandardyouranonyourgeltoestimatethemolecularweightsoftheotherproteinsonyourgel.Asmentionedearlier,theproteinmolecularweightstandardisasetofproteins of knownmolecular weight. Since the rate at which a denatured proteinmoves through apolyacrylamidegelduringelectrophoresisdependsonly in itsmolecularweight, youcanestimate themolecularweightofaproteinbasedonitspositiononthegelinrelationtothemolecularweightstandardproteins. Intheory, themostaccuratewaytodothis is tocreateastandardcurveonsemi-loggraphpaperbyplottingmolecularweightindaltons(Da)orkilodaltons(kDa)vsdistancetraveledonthegelincm.Thedistancetraveledreferstothedistancefromthemiddleoftheproteinbandtothetopoftheresolvinggel(notthebottomofthewellinthestackinggel).

61

Thegraphaboveshowsasamplestandardcurveconsistingof4knownproteins(NOTE–thestandardproteinsonyourgelaredifferent,thisisanexample).Oncethestandardcurveisdrawn,themolecularweightofanyproteinonthegelcanbeestimatedusingthedistanceittraveledongel.Forexample,ifaproteinonthegelpertainingtothisstandardcurvetraveled7.4cm,identify7.4cmontheX-axisandmoveverticallytofindthecorrespondingpositiononthestandardcurve.ThemolecularweightontheY-axis that corresponds to this point on the standard curvewill be your estimate. In this example themolecularweightestimatewouldbe~45,000daltonsor45kDa.Whilethismethodcanbeveryprecise,itsaccuracydependsonthequalityofthedistancemeasurementsandtheprecisionofthebestfitcurve.Asimplermethodtoestimatemolecularweightistocomparetheproteinbandofinteresttotheproteinmolecularweightstandardandvisuallyestimate itsmolecularweight. Forexample, ifyourproteinofinterestispositionedapproximatelyhalfwaybetweenstandardproteinsthatare40kDaand50kDa,thenyouwouldestimate45kDa.Ifthebandisjustaboveandveryclosetothe40kDastandardproteinthenyoumightestimate41or42kDa.Whilethismaynotseemveryprecise,itcangiveyouavalueverycloseto that obtainedusing a standard curve and formost purposes this shouldbe sufficient. In thenextexerciseyouwillestimatetheMWofproteinsusingbothmethodsdescribedabove.

(Dal

tons

or D

a)

62

Exercise3–EstimatingproteinmolecularweightNOTE:A12%polyacrylamidegelcaneffectivelyresolveorseparateproteinsbetween20kDaand100kDa,sobesureNOTtoincludemolecularweightstandardproteinsoutsidethisrangeinyourstandardcurve.

1. Placeyourstainedgelonawhitetransilluminatoranduseasmallrulertomeasureincmthedistancefromthemiddleofthebandtothetopoftheresolvinggelforeachmolecularweightstandardprotein*between20kDaand100kDa.Recordthesevaluesinyournotebook.

2. Plotthesevaluesonthesemi-loggraphpaperfoundattheendofthislabanddrawthebestfitlinethroughthepoints.Acopyofthisstandardcurveshouldbeplacedinyourlabnotebook.

3. Foreachproteinsampleloadedonyourgel,identifythemostprominentband(thiswillbetheproteinofinterest,theotherbandsarecontaminants),measurethedistancefromthemiddleofthebandtothetopoftheresolvinggel.Recordthesevaluesinyournotebook.

4. Estimatethemolecularweightofeachproteinusingthestandardcurveasshownonthepreviouspageandrecordinyournotebook.

5. Estimatethemolecularweightofeachproteinbyvisualcomparisonwiththeproteinmolecularweightstandardsandrecordinyournotebook.

6. Answerthestudyquestionsassociatedwiththislab.

NOTE:Forthislaboratoryyouwillturninaformallabreport.*Theproteinmolecularweightstandardmostlikelyusedonyourgelisshownbelow.Ifadifferentproteinmolecularweightstandardwasusedyourinstructorwillprovidethedetails.

63

64

SDS-PAGELabStudyQuestions NAME:1.SDS-PAGEisanacronymreferringto3specificcomponents(SDS,PAandGE).Indicatewhat

SDS-PAGEstandsforandbrieflydescribeeachofthese3components.2.Whatdoesitmeanto“denature”aproteinandwhyisthisimportantforSDS-PAGE?3.DescribehowyoudenaturedtheproteinsyouusedforSDS-PAGE.4.Whatsubstanceinsampleloadingbufferisresponsibleforbreakingdisulfidebonds?5.Diagramadisulfidebondunder“BEFORE”andalsodiagramwhatitshouldlooklikeafter

boilinginthepresenceofthesubstancefromthepreviousquestion(“AFTER”).

BEFORE AFTER6.Whatisthepurposeofloading“proteinmolecularweightstandards”onthegel?

65

7.Listthemolecularweightsofthestandardproteinsrunonyourgelandchecktheonesthatyouusedtomakeyourstandardcurve.

8.Describeeachofthetwomethodsyouusedtoestimateproteinmolecularweight.9.Whydidyoumeasurethedistanceeachproteintraveledintothegelfromthetopofthe

resolvinggelandnotthebottomofthewell?

66