醋酸纤维素膜电泳分离血浆蛋白(英文版)

Disciplinesinthelaboratory：Mustwearwhitecoatanddon’tbelate.Mustkeepquietwhenyouareinthelaboratory.Don’ttouchanyexperimentalapparatuswithoutmypermission.Anassistantneeded.Collecttheexperimentalreportsandasktwostudentsondutytocleanthelabafterclasseverytime.Theinstructorwillcheckthelaboratoryatlast.Fivescoreswillbelostinyourfinialexaminationifyouwerelateordidnotcleanthelabeachtime.Experimentalreport1.Experimentalprinciple2.Experimentalmaterials(optional)3.Experimentalprocedure4.Resultsandanalysis

orResultsandCalculationIfyougetamembrane(picture),pleasepasteitonthelabnotebook.Describethemembrane(picture)andtrytoexplainthepossiblereasonwhyyougettheresult;Ifyougetthemathematicdata,pleasedosomecalculation.

5.ConclusionUseoneortwosentencestosummarizeyourexperiment.

TheexperimentaltitleSeparationofplasma

proteins

bycelluloseacetatemembraneelectrophoresisElectrophoresisChargedparticlesmovetotheelectrodeofversechargeintheelectricfield.Particles:bio-macromolecule.e.g.DNA,RNAorprotein.Direction:Ifparticlescarrynegativecharge,itwillmovetowardpositive

electrode.Ifparticlescarrypositivecharge,itwillmovetowardelectrode.negativeCelluloseacetatemembrane

Akindofmaterialthathasthenet-likestructure.Thespecialstructurecanretardtheproteinsbythemolecularweight.Proteinlarger,movesslowerintheelectricfield.Proteins

intheplasma

Thousandsofproteinsintheplasma.Themainproteinsarealbumin,

α1-globulin,α2-globulin,β-globulin,fibrinogenand

γ-globulin.Theseproteinscanbeseparatedbycelluloseacetatemembraneelectrophoresis.

1.Experimental

principleThe

isoelectricpoint

(pI),isthe

pH

atwhichaparticular

molecule

carriesnonet

electricalcharge

inthe

environmentalbuffer.

1.Experimental

principle Differentproteinsinplasmahavedifferentisoelectric

points(pI),sotheyhavedifferentquantity

of

electrical

chargeintheelectrophoreticbuffer(pH8.6).Moreover,proteinsalsohavedifferentmolecularweightsandshapes.Underthesameconditionofelectricfield,theywillmoveatthedifferentspeeds,whichiscalledelectrophoreticmobility.Afteraperiodoftime,proteinsinplasmacanbeseparatedbyelectrophoresis.Whataffectselectrophoreticmobility?

External

factors:electric-fieldstrength,pH

valueinthebuffer,ionic

strength,etc.Internalfactors:

quantity

of

electric

charge、molecularweightandshapes.2.Materials(optional)Celluloseacetatemembrane(2×8cm)PowersupplyCoverslip(loadingsample)filterpaperslideglass

tweezerHumanplasmabarbitalbuffer(pH8.6)stainingsolution(Amidoblack10B)Washbuffer3.Experimentalprocedure(1).Soakofmembrane：

Makealoadingsamplelinewithapencilonthenon-reflectivesurface.Thelineshouldbeperpendiculartothelengthofmembraneandatabout1.5cmdistancefromthewidthofmembrane.Then,putthemembraneintotheelectrophoreticbufferandsoakitabout30minutes.

(2).Loadingsample: Pickupamembranewithatweezerandabsorbabundantbufferwithfilterpaper.Useacoversliptodiptheplasmaandloadthesampleontheloadingsamplelineofmembraneonlyonce.(Ifyourepeatloadingsamples,theproteinbandsmaybecurve.)(3).Proteinsseparationbyelectrophoresis:

Putthemembraneoverandclosetosaltbridges.Keepthemembranesurfaceofloadingsamplefacingdown.Letthemembranebeperpendiculartothesaltbridgesandkeeptheendofmembraneneartoloadinglinetightlyatsaltbridgeofnegativeelectrode.

Theprofileofelectrophoretictank1.Saltbridgeofnegativeelectrode;2.Electrophoreticbuffer3.celluloseacetatemembrane4.SaltbridgeofpositiveelectrodeDon’tlettheloadinglinetouchwithsaltbridges. Covertheelectrophoretictankwiththelidandsetthevoltageat120volts,runningabout30~45minutes.(3).Proteinsseparationbyelectrophoresis:(4).Dyingthemembrane: Pickupthemembranewithtweezersanddyetheproteinswithstainingsolution(Amidoblack10B)forabout1~3minutes.(5).Washingthemembrane: Washthestainingmembranewiththewashingbuffer

threetimesintheglassvat.Afterwashing,thedarkblueproteinbandscanbevisualizedonthemembrane.Glassvat#1StainingvatGlassvat#3Glassvat#24.Resultsandanalysis(-)(+)Whythestainingcolorofproteinsaredifferent?Whydoessomeproteinmovefaster?Andwhydoessomeproteinmoveslower?Becausethequantityofalbuministhemostamongtheseproteins,thecolorofalbuministhemostdark.Onthecontrary,thequantitiesofα1-globulinandα2-globulinarerelativelyless,sotheircolorareweakerthanotherproteinbands.

Albuminmovesfastestbecauseitsmolecularweightisthesmallestamongtheseproteins.Onthecontrary,γ-globulinmovesrelativelyslowerbecausethemolecularweightarelargerthanotherproteins.

4.ResultsandanalysisThesignalsofproteinbandsarenotverystrong.Itispossiblethattheloadingsampleislessorstainingtimeisnotenough.Theproteinbandsarenotconsecutivebecausetheloadingsampleisuneven.

4.ResultsandanalysisTheproteinbandslookstooclosesothatsomeproteinsarenotseparatedbycelluloseacetatemembraneelectrophore