Overview Thisprotocolprovidesconsiderationsandgeneralizedproceduresfortheuseofdialysistoalterthecompositionofaproteinsolution.Itisausefulstepinadjustingaproteinsamplefromonebuffertoanother,inadjustingthemetalandsaltionconcentrations,andintheremovalofunwantedsmallmolecules. Procedure BioReagentsandChemicals ProtocolHints. A.PreparationofDialysisTubingChoiceofTubing:1.Dialysistubingisessentiallyacylindricalmembranecontainingpores.Thesizeoftheseporesdeterminesthemolecularweight"cut-off"ofthetubing.Typicalcut-offsare5,000Daltons,10,000Daltons,30,000Daltons,and100,000Daltons.Thus,ifyouchoosetubingwithacut-offof30,000Daltons,allmoleculessmallerthan30kDwillbeabletopassfreelythroughthemembrane(seeHint#1).2.Dialysistubingcanbepurchasedintwoforms:onearrivesasa"dry"rollinaboxwhiletheotheriswetandstoredinaliquidbuffer.Thedryrollmustbepre-treatedbeforeuse.ThisinvolvessoakingthemembraneinddH2O,heatingthemembraneto60°CinBicarbonateSolution,andrinsinginddH2O.Thewetmembranehasalreadybeenpre-treatedforuseandonlyneedstoberinsedinddH2Otoremovepreservatives(seeHint#2).B.UseoftheDialysisTubing1.Afterwashingthetubing(seeProtocolID#113),calculatethelengthoftubingnecessarytocontainthevolumeofyourproteinsampleandcutthetubingtolength,leavinganextrainchortwooneachsideforclosures(seeHint#3).2.Useadialysistubing-specificclosure(e.g.,Spectra/PorClosuresfromSpectrumLaboratories)tocloseoneend,ortietwotightknotsononeendofthetubing(seeHint#4).3.Pipetteyourproteinsampleintothetubingandcloseofftheotherendofthetubingwithanotherclosureorwithtwotightknots.Trytoavoidincludingtoomanyairbubbles(seeHint#5).4.InsertthedialysistubingcontainingyourproteinintoDialysisBufferinalargebeaker.Thevolumeofthebuffershouldbeatleast100timestheoriginalvolumeoftheproteinsample.Thebuffershouldalsobepre-chilledifyourproteinislABIle.5.Stirthebufferslowlywithastirbarandmagneticstirplateforatleast1.5hr.6.DiscardtheDialysisBufferandreplacewiththesamevolumeoffreshDialysisBuffer.7.Dialyzeforanadditional1.5hr(seeHint#6).8.RemovethetubingfromthebeakercontainingDialysisBufferandcarefullyopenoneendofthetubing.Pipettethedialyzedproteinsolutionintotubes. Solutions BicarbonateSolution 2%(w/v)NaH2CO31mMEDTA 
DialysisBuffer MetalandSaltIons(NaCl,KCl,MgCl2,etc.)Othersmallmolecules(Glycerol,DTT,ATP,etc.)TypicalProteinBufferingAgent(Tris,HEPES,Phosphate-based,etc.) SodiumBicarbonateEDTA 1.Chooseyourcut-offwisely(belowthesizeofyourprotein),especiallyifyouhavespentseveraldaysinthecoldroompurifyingyourproteinandareusingdialysisasalastpurificationstep.Otherwise,youwillquicklydiscoverthatyourproteinhasdisappeared.2.Thecontributorofthisprotocolsuggestsusingthewetdialysistubing.However,asthewettubingisusuallymoreexpensivethanthedryroll,thePrincipalInvestigatorshouldbeconsulted.3.Avoidtheintroductionofcontaminantsbywearinggloveswhenhandlingthetubing;otherwise,youmaydiscoverthatyourproteinhasdisappearedorthatyouhavepurifiedhumankeratin.Tubingcanbepurchasedinavarietyofdiameters(orwidths).Thecontributorofthisprotocolsuggeststhatyouimpressyourcolleaguesby"eyeballing"thesufficientlength.AvoidanyattempttoimpressyourPrincipalInvestigatorbyaskingforassistanceinthiscalculation.4.YoucanfillthetubingwithasmallamountofddH2Oandsqueezethetubingfromtheopenendtomakesuretheclosureistight.5.Createasmallproteindialysis"sausage"whenclosingoffthesecondendofthetubing.BECAREFUL,however,toleaveenoughroomforexpansion,especiallyifyourproteinsamplecontainshighsaltconcentrationsandyouaredialyzingagainstabufferthatcontainslowsaltconcentrations.Otherwise,youmaydiscoverthatyourproteinhasdisappearedandyoursausagehasburst.Bubblessmallerthanapproximately250μlarefine.6.TheexchangeofsolutioncomponentsbetweentheproteinsampleandtheDialysisBufferismostefficientifcarriedoutforlongerthan1.5hr.Thisshouldbeempiricallydetermined.However,anovernightincubationisoftenoptimalforremovalofsmallmoleculesfromaproteinsample.
