DuringthisweekyouwillanalyzetheSDSPAGEanddeterminetheproteincontentinthelyophilizedfractionoflipoproteinsgravimetrically.YouwillalsoextractthelipidsfromlipoproteinsaccordingtoBlighandDyer(1959)forfurthercharacterization.Inthenextexperiment(Lipidanalysis),lipidcompositionoftheextractwillbedeterminedbythinlayerchromatography(TLC)followedbyGasliquidchromatography(GLC). Whilequalitativeevaluationcanbedoneonthestainedgelitself,anyquantitativeanalysisisbestdoneonascannedimageofthegel.Usingappropriatesoftware,youcanthencarryouttherequiredanalyses. distancebetweensamplewellandproteinband______________________________________=Rf distancebetweensamplewellanddyefront FurtheranalysisshouldbedoneintheBIOLOGyinstructionalcomputinglaboratory(orathome).Analysisofgel:
Fromthestainedgel,youwillbeabletogetinformationabout:Purificationprocess
Thisshouldbeobviousbyvisualinspection.YoushouldseethreecharacteristicbandsofapoLp-I,apoLp-II,andapoLp-IIIinyourpooledlipophorinfraction.Hopefully,thesebandspeakinthefractionsthatyoupooled.Other,adjacentfractionsmaycontainthesethreebands,butyoushouldnotseealloftheseatdensitiesthataremuchhigher.Purityoflipophorin
Fromthescannedimage,youcanestimatethepurityofyourpooledlipophorinfractionifyouassumethatallproteinsarestainedbyCoomassieBrilliantBluewithsimilarintensity(thisisnotnecessarilytrue,however).Dividingthedensitiesofthethreeapoproteinsbythecombineddensitiesofallbandsinthatlanewillresultinthepercentageoflipophorin.Molecularweightofapoproteins
TheelectrophoreticmobilityofaproteininanSDSgelisequivalenttoitsmolecularweight.Hence,wecandeterminethesizeofthethreeapolipophorinmoleculesifwegenerateastandardcurvefromknownmolecularweightMarkers.ThisisaccomplishedbyplottingtheirRfvaluesagainstthelogoftheirmolecularweights(Mr).Apoproteinratio
Fromaquantitativeanalysisofthegelscan,youcandeterminethemolarratiobetweentheapoproteins,againassumingthateachbindsCoomassieBrilliantBluesimilarly.Thedensityofeachbandsisthenproportionaltotheamountofproteininit.Inordertogetinformationaboutthemolarratio,youhavetodivideeachdensityreADIngbythemolecularweightoftheapoprotein,asdeterminedbefore;theratiobetweentheresultingvaluesrepresentsthemolarratio.Scanning
Firstyoumustscanthestainedanddestainedgel.Placethegelbetweentwoclearplasticsheets,andmakesurethatnodestainingsolutionleaksfromthesandwich.GototheMacintheadjacentcomputerlabandstartthe"DeskScan"software.Imageanalysis
GelanalysisisbestcarriedoutwiththeNIHImageprogram.ThisprogramwasdevelopedbytheNationalInstituteofHealth.InformationaboutitsusecanbeobtainedfromtheNIHWebsite.Sincetheprogramisfree,youcanalsodownloaditontoyourownMac,ifyouhaveone.AfreePCversionofImage,calledScionImageforWindows,isavailablefromScionCorporation.(ThereisalsoImage/J,aJavaprograminspiredbyImage).Forouranalysisweneedtheprogramandthegelscanningmodule.ThissoftwareisloadedontotheMACSintheInstructionalComputingroom.
