使用ImageJ分析Western.docx

使用ImageJ分析Western.docx

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使用ImageJ分析Western

使用ImageJ分析Western Blot

（2013-04-1621:

40:

31）

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image

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western

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ThefollowinginformationisanupdatedversionofamethodforusingImageJtoanalyzewesternblotsfromanow-deprecatedolderpage.Don’tusethealternatemethodsdiscussedontheoldpage,astheyaresubjecttowaytoomuchuserbias.

Apdfcopyofthispageisavailable.

ImageJ（http:

//rsb.info.nih.gov/ij/index.html）canbeusedtocomparethedensity（akaintensity）ofbandsonanagargelorwesternblot.Thistutorialassumesthatyouhavecarriedyourgelorblotthroughthevisualizationstep,sothatyouhaveadigitalimageofyourgelin.tif,.jpg,.pngorotherimageformats（.tifwouldbethepreferredformattoretainthemaximumamountofinformationintheoriginalimage）.Ifyouarescanningx-rayfilmonaflatbedscanner,makesureyouuseascannerwiththeabilitytoscantransparencies（i.e.film）.Seethereferencesattheendofthistutorialforadiscussionofthevariouswaysthatyoucanscrewthisstepup.

ThemethodoutlinedhereusestheGelAnalysismethodoutlinedintheImageJdocumentation:

GelAnalysis.YoumayprefertouseitinsteadofthemethodsIoutlinebelow.Thereshouldbeverylittledifferencebetweentheresultsobtainedfromthevariousmethods.ThisversionofthetutorialwascreatedusingImageJ1.42qonaWindows764-bitinstall.

1.OpentheimagefileusingFile>OpeninImageJ.

2.Thegelanalysisroutinerequirestheimagetobeagray-scaleimage.ThesimplestmethodtoconverttograyscaleistogotoImage>Type>8-bit.YourimageshouldlooklikeFigure1.

3.ChoosetheRectangularSelectionstoolfromtheImageJtoolbar.Drawarectanglearoundthefirstlane.ImageJassumesthatyourlanesrunvertically（soindividualbandsarehorizontal）,soyourrectangleshouldbetallandnarrowtoencloseasinglelane.Ifyoudrawarectanglethatisshortandwide,ImageJwillswitchtoassumingthelanesrunhorizontally（individualbandsarevertical）,leadingtomuchconfusion.

4.Afterdrawingtherectangleoveryourfirstlane,pressthe1keyorgotoAnalyze>Gels>SelectFirstLanetosettherectangleinplace.The1stlanewillnowbehighlightedandhavea1inthemiddleofit.

5.Useyourmousetoclickandholdinthemiddleoftherectangleonthe1stlaneanddragitovertothenextlane.Youcanalsousethearrowkeystomovetherectangle,thoughthisisslower.Centertherectangleoverthelaneleft-to-right,butdon’tworryaboutliningitupperfectlyonthesameverticalaxis.Image-Jwillautomaticallyaligntherectangleonthesameverticalaxisasthe1strectangleinthenextstep.

6.Press2orgotoAnalyze>Gels>SelectNextLanetosettherectangleinplaceoverthe2ndlane.A2willappearinthelanewhentherectangleisplaced.

7.RepeatSteps5+6foreachsubsequentlaneonthegel,pressing2eachtimetosettherectangleinplace（Figure3）.

8.Afteryouhavesettherectangleinplaceonthelastlane（bypressing2）,press3,orgotoAnalyze>Gels>PlotLanestodrawaprofileplotofeachlane.

9.Theprofileplotrepresentstherelativedensityofthecontentsoftherectangleovereachlane.Therectanglesarearrangedtoptobottomontheprofileplot.Intheexamplewesternblotimage,thepeaksintheprofileplot（Figure4）correspondtothedarkbandsintheoriginalimage（Figure3）.Becausetherewerefourlanesselected,therearefoursectionsintheprofileplot.Higherpeaksrepresentdarkerbands.Widerpeaksrepresentbandsthatcoverawidersizerangeontheoriginalgel.

10.Imagesofrealgelsorwesternblotswillalwayshavesomebackgroundsignal,sothepeaksdon’treachdowntothebaselineoftheprofileplot.Figure5showsapeakfromarealblotwheretherewassomebackgroundnoise,sothepeakappearstofloatabovethebaselineoftheprofileplot.Itwillbenecessarytocloseoffthepeaksothatwecanmeasureitssize.

11.ChoosetheStraightLineselectiontoolfromtheImageJtoolbar（Figure6）.Foreachpeakyouwanttoanalyzeintheprofileplot,drawalineacrossthebaseofthepeaktoenclosethepeak（Figure5）.Thissteprequiressomesubjectivejudgmentonyourparttodecidewherethepeakendsandthebackgroundnoisebegins.

12.Notethatifyouhavemanylaneshighlighted,thelaterlaneswillbehiddenatthebottomoftheprofileplotwindow.Toseetheselanes,pressandholdthespacebar,andusethemousetoclickanddragtheprofileplotupwards.

13.WheneachpeakhasbeenclosedoffatthebasewiththeStraightLineselectiontool,selecttheWandtoolfromtheImageJtoolbar（Figure8）.

14.Usingthespacebarandmouse,dragtheprofileplotbackdownuntilyouarebackatthefirstlane.WiththeWandtool,clickinsidethepeak（Figure9）.Repeatthisforeachpeakasyougodowntheprofileplot.Foreachpeakthatyouhighlight,measurementsshouldpopupintheResultswindowthatappears.

15.Whenallofthepeakshavebeenhighlighted,gotoAnalyze>Gels>LabelPeaks.Thislabelseachpeakwithitssize,expressedasapercentageofthetotalsizeofallofthehighlightedpeaks.

16.ThevaluesfromtheResultswindow（Figure10）canbemovedtoaspreadsheetprogrambyselectingEdit>CopyAllintheResultswindow.Pastethevaluesintoaspreadsheet.

Note:

IfyouaccidentallyclickinthewrongplacewiththeWand,theprogramstillrecordsthatclickedareaasapeak,anditwillfactorintothetotalareausedtocalculatethepercentagevalues.Obviouslythiswillskewyourresultsifyouclickinareasthataren’tpeaks.Ifyoudohappentoclickinthewrongplace,simplegotoAnalyze>Gel>LabelPeakstoplotthecurrentresults,whichdisplaystheincorrectvalues,butmoreimportantlyresetsthecounterfortheResultswindow.Gobacktotheprofileplotandbeginclickinginsidethepeaksagain,startingwiththe1stpeakofinterest.TheResultswindowshouldclearandbeginshowingyournewvalues.Whenyou’resureyou’veclickinallofthecorrectpeakswithoutaccidentallyclickinginanywrongareas,youcangobacktoAnalyze>Gels>LabelPeaksandgetthecorrectresults.

Dataanalysis

Withyourdatapastedintoaspreadsheet,youcannowcalculatetherelativedensityofthepeaks.Asareminder,thevaluescalculatedbyImageJareessentiallyarbitrarynumbers,theyonlyhavemeaningwithinthecontextofthesetofpeaksthatyouselectedonthesinglegelimageyou’vebeenworkingon.Theydonothaveunitsofμgofproteinoranyotherreal-worldunitsthatyoucanthinkof.Thenormalprocedureistoexpressthedensityoftheselectedbandsrelativetosomestandardbandthatyoualsoselectedduringthisprocess.

1.Placeyourdatainaspreadsheet.Oneofthepeaksshouldbeyourstandard.Inthisexamplewe’llusethe1stpeakasthestandard.

2.InanewcolumnnexttothePercentcolumn,dividethePercentvalueforeachsamplebythePercentvalueforthestandard（the1stpeakinthiscase,26.666）.

3.Theresultingcolumnofvaluesisameasureoftherelativedensityofeachpeak,comparedtothestandard,whichwillobviouslyhavearelativedensityof1.

4.Inthisexample,the2ndlanehasahigherRelativeDensity（1.86）,whichcorrespondswellwiththesizeanddarknessofthatbandintheoriginalimage（Figure1）.Recallthatthesedataarefortheupperrowofbandsontheoriginalwesternblotimage.

5.Ifyouwanttocomparethedensityofsamplesonmultiplegelsorblots,youwillneedtousethesamestandardsampleoneverygeltoprovideacommonreferencewhenyoucalculateRelativeDensityvalues.Seethesectionsbelowformoredetaileddiscussionoftheserequirements.

6.Inordertotestforsignificantdifferencesbetweentreatmentsinanexperiment,allofyourgelsorblotswillneedtobescannedandquantifiedusingthismethod,andthevalueswillbeexpressedintermsofRelativeDensity,oryoucantreatRelativeDensityasafold-changevalue（i.e.aRelativeDensitydifferenceof2betweenacontrolandtreatmentwouldindicatea2-foldchangeinexpression）.Ifyouwillbeusinganalysisofvariancetechniquestotestyourdata,youmayneedtoensurethatyourRelativeDensityvaluesarenormallydistributedandthatthereishomogeneityofvarianceamongthedifferenttreatments.

7.Itshouldbenotedherethatsomeresearchersmaketheextraefforttoincludeasetofserialdilutionsofaknownstandardoneachblot.Usingtheserialdilutioncurveandthequantificationtechniquesoutlinedabove,itshouldbepossibletoexpressyoursamplebandsintermsofpicogramsornanogramsofprotein.

Amoreinvolvedexampleusingloading-controls.

We’lluseFigure12asarepresentativewesternblot.Onthisblot,wewillpretendthatweloadedfourreplicatesamplesofprotein（fourpipetteloadsoutofthesamevialofhomogenate）,soweexpectthedensitiesineachlanetobeequivalent.Theupperrowofbarswillrepresentourproteinofinterest.Thelowersetofbarswillrepresentourloading-controlprotein,whichismeanttoensurethatanequalamountoftotalproteinwasloadedineachlane.Thisloading-controlproteinisaproteinthatispresumablyexpressedataconstantlevelregardlessofthetreatmentappliedtotheoriginalorganisms,suchasactin（thoughmanypeoplewillquestiontheassertionthatactinwillbeexpressedequivalentlyacrosstreatments）.

LookingatFigure12,wehadhopedtoloadequivalentamountsoftotalproteinineachlane,butafterrunningthewesternblot,thesizeandintensityofthelowerbarsineachlanevariesquitealot.Thetwoleftlanesappearequivalent,butthe3rdlanehashalfthedensity（grayvalue）comparedtolanes1+2,whilelane4hash