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1、WesternBlot图片灰度分析–PhotoshopComparingtheintensityofbandsonaWesternblotcanbedoneinanumberofwaysusingsoftwarethatiscommonlyfoundonlabcomputersorfreelyavailablefordownload.Thefollowingdocumentoutlinessomeofthosemethods.Foralongtime,thede-factostandardforanalysisinlabsthatdidn'twanttospringfo
2、rmulti-thousand$$commercialdensitometrysoftwarewasAdobePhotoshoporoneofthecompetingphotoeditingprograms.Allyoureallyneedinaprogramisafreehandselectiontoolandawaytomeasurethemeangrayvalueinsidetheselection.We'llstartwithAdobePhotoshopasanexample(youcanfindmanyreferencesintheliteraturethat
3、includephrasessuchas"densitometrywascarriedoutinPhotoshop"inthemethodssection).Tostartwith,you'llneedtoscaninyourxrayfilmonaflat-bedscanner.Thiscanbeacheapconsumerunit,oramoreexpensivetransparencyscannerifyouhaveaccesstosuchabeast.Youcanscanthefilmasagrayscaleimage,andsettheresolutiontoa
4、mediumvalue(300-400dpi).1.OpenthescannedimageinPhotoshop.2.UnderImage>Mode,checkthegrayscaleoptionifit'snotalreadyselected.Wedon'tcareaboutcolorinformation,onlygrayscaleinformation,sowecandiscardthecolorinformation.1.UnderImage>Adjustments,selectInvert(orpressCtrl+I).Nowthedarkpartsofthe
5、filmarelight,andthelightpartsaredark.Thisisusefullater,asthehigh-expressionbands,whicharedarkonthefilm,willhavehighnumericalvalueswhenwemeasurethem.Whenphotoprogramsreportdarkness/lightnessvalues,thedarkpointshavevaluesnearzero,andthelightpointshavevaluesthatmaxoutat255.Theinvertedscan2.
6、Choosethelassotoolfromthetoolpalette.3.Onthefirstband,usethelassotooltodrawalineallthewayaroundtheedgesofthefirstband.Thisiswhereyourjudgementcomesintoplay,determiningwheretheedgesofthebandare,andwhatissimplybackground.6.ForPhotoshopCS2/3:Ifthehistogramwindowisnotopenbydefault,gotoWindow
7、>Histogramtoopenthehistogram.InnewerversionsofPhotoshop(CS2,CS3)thereisasmallarrowinacircleintheupperrightcornerofthehistogramwindow.Clickonthisandchoose'expandedview'toshowthevaluesforyourselection.ForPhotoshopv.5+6:GotoImage>Histogramtodisplaythehistogramforthecurrentse
