Asinsectshaveanopencirculatorysystem,thehemolymphcanbesimplycollectedthroughanincisioninthebodywall.Mostconveniently,youshouldcutalegandlettheblooddripintoachilledglassbeaker.Togetmaximumyield,itisnecessarytoflushoutthebloodbyinjectingbufferintothebodycavity.Theexperimentalprocedureisschematicallyshowninthefollowingfigure. Followingtheremovalofhemocytes,potassiumbromideisaddedtothehemolymphtoforma44%solution.Thisisoverlayeredwithphosphatebufferedsaline(PBS),andcentrifugedtoformadensitygrADIent.Eventually,allproteinsfloatorsinktothezonethatcorrespondstotheirdensity.Havingalowerdensity,theyellowcoloredlipoproteinfloatshigherthannon-lipoproteins,includingagreencyanoprotein.Theseparatedproteinsarefractionated.Fortheindividualfractions,thedensityisdeterminedbyrefractometry,proteincontentandcompositionbytheBradfordassayandelectrophoresis,respectively,andthelipidcompositioncanbeanalyzedbythinlayerandgaschromatography. Note:Youneedtowearglovesthroughouttheprocedure. Eachgroupstakes10-15locusts. 1.Take2-5mlofPBSinaclean100mlbeakerandadd25µlof200mMPMSFHighlypoisonous! 2.Tobleedholdthelocustsbyalllegsandwingsandcutoffalegnearthorax(seepicture).Makesurenoinsectsalivadripsintothebeaker(haveKleenexreadytowipeoffsaliva). 3.GentlyinjectafewhundredmicrolitresofPBSthroughsegmentssubcutaneously.Donotinjurethegut!. 4.Flushhemolymph(yellowishduetothepresenceofcarotin)carefullyoutbysqueezingthethoraxareagently.Collectbloodfrom10-12locusts. 5.Putthebledlocustsinajar.Whenfinishedbleeding,movethejartothefreezer.Disposeoffthedeadanimalsatalaterdate. 6.Makeupthehemolymphsolutionto~15mlwithPBS. 7.Add8.9gofKBrcrystalsandstirgentlyusingacleanstirbar. 8.Transferthedissolvedportionintoa50mlmeasuringcylinderandaddmore(2-3ml)PBStotheundissolvedKBrandstiruntildissolved.Poolthesesolutionsandmakeupto20mlwithPBSandstirfurtherinthemeasuringcylinder. 9.Transfersolutionintoa30mlplasticsyringe(heldbyaclamp)attachedtoa18gaugeneedleandacapillarytubing(seepicture). 10.Letthehemolymphflowintotheheatsealablecentrifugetube. 11.Addanother20mlPBSintothesyringeandoverlayonthehemolymph. 12.Turnontheheatsealertowarmitup. 13.Balanceagainstthesamplefromtheothergroup;youmustbeexact!UsetheanalyticalbalanceandaddorremovePBStoorfromthetubes.Ifliquidisintheneck,removewiththetipofatissue. 14.Placethemetalcapofthetubes,andsealwiththeheatsealer.Assoonasthecapreachestheshoulderofthetube,quiclymovethetubetotherightandpushthecapdownwiththeheatsink. 15.PlacethefourtubesintheVTi50rotorintheoppositepositionsandplacetherotorintheultracentrifuge. 16.Makesurethelidisclosed.Turnonthevacuumofthecentrifuge.Settherunconditionsto10C,brakeon,speed45,000,time4.5h. 17.Spinat45,000rpmfor4.5h,andleavesampleovernightinthecentrifuge.. 1.Aftertherunisover,turnthevacuumoffandwaituntilthechamberpressurereachesatmosphericpressure.Nowyoucanremovethetubes. 2.Noteanyunusualobservationsduringorafterthecoentrifugation(e.g.,leaks),andrecordinthecentrifugebook.Cleanthechamberandtherotor,andstartaDRYRUNtoavoidcondensationinthecentrifuge. 3.Usingasharpsingleedgedrazorcarefullycutopencentrifugetube. 5.Determinedensityusingtherefractometer. 6.AlsodetermineUVabsorbanceat280nm.RemembertouseUV-permeableplasticcuvettes(1pair). 7.Whiledeterminingtherefractiveindexplace2piecesofdialysistubing(eachapprox.20cmlong)intoa500mlbeakerfilledwithdist.water. 8.Leaveanaliquot(300µl)ofeveryotherfractioninlabeledmodifiedEppendorftubesfordialysis. 9.Dialyzeagainst~1lofdist.waterinthecoldroom,changingthebufferonce. 10.Carefullytransfertheyellowlipophorinfractions(normally3-5fractions)intodialysistubing(closedoffatthebottomwithanorangeclamp)usingapasteurPipette. 11.Closethedialysistube,makingsuretoleaveenoughroomforexpansionduringthedialysis. 12.Dialyzeagainst4lofdist.waterinthecoldroomovernight.Changewaterandrepeatdialysisatleasttwice.
ExperimentalProtocol
(about2-3hnon-continuous)
4.SetuptheBioRadfractioncollector.Fractionatecontentinto20twomilliliterfractions.
