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1、WesternBlot图片灰度分析–PhotoshopComparingtheintensityofbandsonaWesternblotcanbedoneinanumberofwaysusingsoftwarethatiscommonlyfoundonlabcomputersorfreelyavailablefordownload.Thefollowingdocumentoutlinessomeofthosemethods.Foralongtime,thede-factostandardforan
2、alysisinlabsthatdidn'twanttospringformulti-thousand$$commercialdensitometrysoftwarewasAdobePhotoshoporoneofthecompetingphotoeditingprograms.Allyoureallyneedinaprogramisafreehandselectiontoolandawaytomeasurethemeangrayvalueinsidetheselection.We'llstartw
3、ithAdobePhotoshopasanexample(youcanfindmanyreferencesintheliteraturethatincludephrasessuchas"densitometrywascarriedoutinPhotoshop"inthemethodssection).Tostartwith,you'llneedtoscaninyourxrayfilmonaflat-bedscanner.Thiscanbeacheapconsumerunit,oramoreexpen
4、sivetransparencyscannerifyouhaveaccesstosuchabeast.Youcanscanthefilmasagrayscaleimage,andsettheresolutiontoamediumvalue(300-400dpi).1.OpenthescannedimageinPhotoshop.2.UnderImage>Mode,checkthegrayscaleoptionifit'snotalreadyselected.Wedon'tcareaboutcolor
5、information,onlygrayscaleinformation,sowecandiscardthecolorinformation.1.UnderImage>Adjustments,selectInvert(orpressCtrl+I).Nowthedarkpartsofthefilmarelight,andthelightpartsaredark.Thisisusefullater,asthehigh-expressionbands,whicharedarkonthefilm,willh
6、avehighnumericalvalueswhenwemeasurethem.Whenphotoprogramsreportdarkness/lightnessvalues,thedarkpointshavevaluesnearzero,andthelightpointshavevaluesthatmaxoutat255.Theinvertedscan2.Choosethelassotoolfromthetoolpalette.3.Onthefirstband,usethelassotooltod
7、rawalineallthewayaroundtheedgesofthefirstband.Thisiswhereyourjudgementcomesintoplay,determiningwheretheedgesofthebandare,andwhatissimplybackground.6.ForPhotoshopCS2/3:Ifthehistogramwindowisnotopenbydefault,gotoWindow>Histogramtoopenthehistogram.Innewer
8、versionsofPhotoshop(CS2,CS3)thereisasmallarrowinacircleintheupperrightcornerofthehistogramwindow.Clickonthisandchoose'expandedview'toshowthevaluesforyourselection.ForPhotoshopv.5+6:GotoImage>Histogramtodisplaythehistogramforthecurrentse
