ProteinPurification

HDexon1,16,38,56,and82CAGsfusedwithGST.

Makingconstructs.

ExonIofHDgenewasamplifiedfrompartialCDNAconstructsobtainedfromnormal(16CAGs)andpatient(48CAGs).TheprimerswereengineeredwithrestrictionenzymeBamHIsiteinthesenseprimerandEcoRIintheantisenseprimer.theprimersequencesare:sense,5"CGG CCC CAG GGA TCC GGG GAC TGC 3"; and antisense, 5" TGC GGC TGA GGC AGA ATT CGG GTG TCG 3". digested PCR products were ligated directionally to corresponding digested bacterial expession vector, pGEX2T (4.9 kb) after phosphatase treatment of the vector with calf intestine alkaline phosphatase. Transformation of ligated products was performed by using Top 10 (Invitrogen,Inc)competentcells,HDexonIinsertbothfornormal(16CAG),expanded(48CAG)andotherrepeatsizeswerecheckedbydigestingwithBamHIandEcoRIrestrictionenzymesandelectrophoresisaswellasbysequenceanalysis.

ExpressionofGSTfusionprotein.

Grow5mlofovernightcultureat37°CusingLBbroth(with100mg/literAmicillin).NextdayaddovernightculturetofreshLB(ampinit)at1:20ratioandtillODreaches0.6-1.0atfixedwavelengthA600.

Induceexpressionoffusionproteinsbyaddingisopropyl-B-D-thiogalactoside(IPTG)toafinalconcentrationof1.0mM.Allowthecellstogrowforanadditional3hoursat37°C.Setuptwotubesforeachcolony/construct,onewithIPTGandtheotheronewithoutIPTG.

Take1mlofcellsandspindownfromallsamplesbothforinducedanduninduced.Tothecellpelletadd100µlreducingsolution.Boilcellswithreducingsol.for5minutesandsonicateandload10µlofbothinduced(plusIPTG)anduninduced(minusIPTG).

Note:Theconstructsp16,p38,p56andp82arecheckedforexpressionandpurifiedprotein:foradditionalpurificationlikeanalyticalgelfiltrationandotherpurposestheseconstructsarereadytouse.Expressionisgoodat3hoursIPTGinduction.

PurificationofGSTfusionprotein.

Growa5mlcultureofcellsHDgeneinLB(containing100mg/litreampicillin)at37°C.Usethiscultureinoculateandexpandtheculture.Inoculate1litreofLBbroth(containing100mg/1liter)with100mlofcellculture(1:10cultureandLBdilution).GrowthecellstillODreaches0.6-1.0at600nmfixedwavelength.InducecellswithIPTGfinalconcentrationof1mMfor3hours(at3hoursyougetgoodexpression).

Spindowncellsfor15minutesat2000RPMandwashcellsthreetimeswith1XPBS.Keepcellsoniceatalltimes.Add10mllysissol.(containingproteaseinhibitorsAprotonin1%from1000xandAEBSF1mM).Sonicatecellsfor45secondsforthreetimes.AtthisstageyoucanseelightbrowntransIlluminantsol.AddtritonX(1%finalconcentration).Placetubesinrotaryshakerat4°Cfor15minutes.Spincellsfor15minutesat7000RPM,collectsupernatantintoBeckmancentrifugetube.Spinagainfor30minutesat45K.Separatesupernatant(ourproteinissoluble).

Add2mlof50%gluthionesepharosebeads(fromPharmacia)tothelysedcells,incubateat4°Cfor5hoursorovernightonarotator.

Spinbeadsandseparatesupernatant(atthisstagetargetproteinbindtosepharosebeads).Washbeads3timeswith50volumesof1XPBS(containing1%triton).Washoncewith50volumesof50mMTris(pH7.5)and150mMNaCl.

Eluteproteinfrombeadsusing3-4mlsof10mMreducedgluthionein50mMTris(pH8.0).Eluteagain1-2mlsofthe10mMgluthione.Dialyseproteinindialysisbufferfor5-8preferablyovernight.Runthedialysedproteinon10%SDSPAGEgeltoverifysizeandpurificationprocedure.

Reducingsolution.

• pinchbromophenolblue
• 2.5ml0.5MTrispH6.8
• 2mlglycerol
• 2ml10%SDS
• 2.5mlddH₂O
• 1mlbeta-mercaptoethanol

Lysisbuffer.

• 1XPBS
• 100mMEDTA
• 1%from1000xapropotin
• 1mMAEBSF
• 0.5mMDTT

Dialysisbuffer.

• 20mMHepes
• 150mMKCL
• 0.2mMEDTA
• 1mMAEBSF
• 20%glycerol

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