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1、WesternBlot图片灰度分析-ImageJThismethodistheGelAnalysismethodoutlinedintheImageJdocumentation:GelAnalysis.Youmayprefertouseitinsteadofthemethodsoutlinedbelow.Therewilllikelybeverylittledifferenceintheresultsbetweenthevariousmethods1.Openyourfile.2.GotoAnalyze>Ge
2、ls>GelAnalyzerOptionsandclicktheboxesforLabelWithPercentages,OutlineLanesandInvertPeaks.3.ChoosetheRectangularSelectiontool.Drawarectanglearoundyourfirstlane.Encompasssomeareaofthelaneaboveandbelowthebandofinterest.EditJuly2009:Notethatforagelwiththelanesor
3、ientedverticallyasshownhere(i.e.thevisiblebandsarehorizontalacrosstheimage),youwanttomakeyourboundingrectangletallerthanitiswide.However,ifyouhavetheimagerotatedsothatthelanesarerunninghorizontally,youneedtomakeyourboundingrectangleatleasttwiceaswideasitist
4、all,atwhichpointImage-Jwillrecognizethatyourlanesarehorizontalanditwillallowyoutomovetheboxupordowntheimagetoenclosetheneighboringlanes.4.Pressthe1button(orgotoAnalyze>Gels>SelectFirstLane).Anewwindowwillpopupwithacopyofyourimageandalabeloveryourfirstrectan
5、gularselection.5.Usethearrowkeystomovetherectangleoverthenextlane.Press2(orgotoAnalyze>Gels>SelectNextLane)toplaceaselectionaroundthelane.Repeatthisforeachlaneonthemembrane,movingtheboxandpressing2toplacetheselection.6.Whenfinished,press3(orgotoAnalyze>Gels
6、>PlotLanes),whichpopsupanewwindowwithaprofileplotofeachlane.1.NowchoosetheStraightLineselectiontool.Atthebaseofeachpeak,drawalinefromonesideofthepeaktotheother.Thisenclosestheareaofthepeak.Thetailstoeithersideofthepeakarethebackgroundsignal.Notethatifyouhav
7、emanylanes,thelaterlaneswillbehiddenatthebottomoftheprofileplot.Toseetheselanes,pressandholdthespacebar,andusethemousetodragtheprofileplotupwards.2.WheneachpeakhasbeenclosedoffatthebasewiththeStraightLinetool,choosetheMagicWand(Wandtracingtool)fromthetoolpa
8、lette.3.Usingthespacebarandmouse,dragtheprofileplotbackdownuntilyouareatthetoppeak.Withthewand,clickinsidethepeak.Repeatthisforeachpeakasyougodowntheprofileplot.4.Wheneachpeakhasbeenselected,gotoAnalyz
