Lipoprotein Analysis week 3相关交流-蚂蚁淘在线

DuringthisweekyouwillanalyzetheSDSPAGEanddeterminetheproteincontentinthelyophilizedfractionoflipoproteinsgravimetrically.YouwillalsoextractthelipidsfromlipoproteinsaccordingtoBlighandDyer(1959)forfurthercharacterization.Inthenextexperiment(Lipidanalysis),lipidcompositionoftheextractwillbedeterminedbythinlayerchromatography(TLC)followedbyGasliquidchromatography(GLC).

Analysisofgel:

Fromthestainedgel,youwillbeabletogetinformationabout:

theseparationduringultracentrifugation

thepurityofyourlipophorinfraction

themolecularweightoftheapolipoproteins

themolarratioofthethreeapoproteinsinthelipophorinmolecule

Whilequalitativeevaluationcanbedoneonthestainedgelitself,anyquantitativeanalysisisbestdoneonascannedimageofthegel.Usingappropriatesoftware,youcanthencarryouttherequiredanalyses.

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).

distancebetweensamplewellandproteinband______________________________________=Rf

distancebetweensamplewellanddyefront

Apoproteinratio

Fromaquantitativeanalysisofthegelscan,youcandeterminethemolarratiobetweentheapoproteins,againassumingthateachbindsCoomassieBrilliantBluesimilarly.Thedensityofeachbandsisthenproportionaltotheamountofproteininit.Inordertogetinformationaboutthemolarratio,youhavetodivideeachdensityreADIngbythemolecularweightoftheapoprotein,asdeterminedbefore;theratiobetweentheresultingvaluesrepresentsthemolarratio.

Scanning

Firstyoumustscanthestainedanddestainedgel.Placethegelbetweentwoclearplasticsheets,andmakesurethatnodestainingsolutionleaksfromthesandwich.GototheMacintheadjacentcomputerlabandstartthe"DeskScan"software.

Pre-scanthegelasgreyscalepicture.

Selecttheareayouneedtoscan(=theentiregel).

ScanthegelandsavethefileasaTIFFfile.

TransfertheTIFFfiletoadisk.

FurtheranalysisshouldbedoneintheBIOLOGyinstructionalcomputinglaboratory(orathome).

Imageanalysis

GelanalysisisbestcarriedoutwiththeNIHImageprogram.ThisprogramwasdevelopedbytheNationalInstituteofHealth.InformationaboutitsusecanbeobtainedfromtheNIHWebsite.Sincetheprogramisfree,youcanalsodownloaditontoyourownMac,ifyouhaveone.AfreePCversionofImage,calledScionImageforWindows,isavailablefromScionCorporation.(ThereisalsoImage/J,aJavaprograminspiredbyImage).Forouranalysisweneedtheprogramandthegelscanningmodule.ThissoftwareisloadedontotheMACSintheInstructionalComputingroom.

StarttheImageprogram.

OpenyourTIFFfilefromyourdisk.DetailedinstructioncanbefoundintheImagemanual.

IftheSetuptoplotGelandPlotlanecommandsarenotshownintheSpecialmenu,usetheLoadMacrostoopenthemacroscontainedinthefileGelPlottingMacros.

Closeallimagewindowsexceptforthegeltobeanalyzed.

Dataselection:

Usetherectangularselectiontooltooutlinethefirstlane.Thisistheleftmostlaneforverticallyorientedlanesandthetoplaneforhorizontallanes.

SelectMarkFirstLaneintheSpecialmenu.Acopyoftheimagewillbedisplayedwiththefirstlaneoutlined.

Movetherectangularselection(byclickinginsideitanddragging)andoutline(usingMarkNextLane)eachoftheotherlanesinsuccession.SelecttheMarkerlane,thepooledlipophorinlane,andoneortwoofyourlipophorincontainingfractions.

UsePlotLanestogeneratethelaneprofileplots.

Molecularweightdetermination:

Inthemarkerlane,identifythemajorbandsandtheirmolecularweights.

Measurethedistanceforeachband.

Drawasemilogplotofmolecularweightsvsrelativemobility(Rf)forthemarkerlanes.Usethisestimatethemolecularweightsofprominentbands.

Quantitation:

Usethelinedrawingtooltodrawbaselinesanddroplinessothateachpeakdefinesaclosedareaasshownabove.Notethatyoucanholdtheshiftkeydowntoconstrainlinestobevertical.

Measuretheareasofthepeaksbyclickinginsideeachoneinsuccessionwiththewandtool.

Option-clickwiththetexttooltoautomaticallylabelthepeaks,inreverseorder,withtheareameasurements.Theareameasurementsarealsorecordedintabularform,andcanbedisplayed(ShowResults)printed(Print)orexported(Export)toaspreadsheet.

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Lipoprotein&nbsp;Analysis&nbsp;week&nbsp;3