a in-solution digestion protocol：在溶液中消化的协议

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1、AnIn-GelDigestionProtocolThisprotocoldescribesthedigestionofaproteinpresentinanSDS-PAGEgelbandwithtrypsin.Thebandcanbetakenfromeithera1Dor2Delectrophoresisgel.ReagentsAllreagentsarepreparedimmediatelypriortouse.Thewaterusedinallcomponentsoftheprocedureis17megaohm/cmNanopure®-orMilliQ®-typewa

2、ter.TheacetonitrileandmethanolareHPLCgrade.Allotherreagentsarethehighestpossiblecommercialgradeavailable.Thetrypsinusedisasequencing-gradeenzymethathasbeenmodifiedtoinhibitautolysisandtominimizenon-trypticproteaseactivities.Thereagentsarepreparedineither20-mLglassvialswithTeflonlinedcapsor1.

3、5-mLplasticmicrocentrifugetubes.1.Washsolution:Add10mLofmethanolto5mLofwater.Add1mLofaceticacidandadjustthetotalvolumeto20mLwithwater.Thefinalconcentrationsare50%(v/v)methanoland5%(v/v)aceticacid.2.100mMammoniumbicarbonate:Dissolve0.2gofammoniumbicarbonatein20mLofwater.3.50mMammoniumbicarbon

4、ate:Mix2mLofthe100mMammoniumbicarbonatewith2mLofwater.4.10mMDTT:Place1.5mgofdithiothreitolina1.5-mLplasticcentrifugetube.Add1.0mLof100mMammoniumbicarbonateanddissolvethedithiothreitol.5.100mMiodoacetamide:Place18mgofiodoacetamideina1.5-mLplasticcentrifugetube.Add1.0mLof100mMammoniumbicarbona

5、teanddissolvetheiodoacetamide.6.Trypsinsolution:Add1.0mLoficecold50mMammoniumbicarbonateto20µgofsequencing-grademodifiedtrypsin.Dissolvethetrypsinbydrawingthesolutionintoandoutofthepipette.Keepthetrypsinsolutiononiceuntiluse.Thefinalconcentrationis20ng/µLtrypsin.7.Extractionbuffer:Add10mLofa

6、cetonitrileto5mLofwater.Add1mLofformicacidandadjustthetotalvolumeto20mLwithwater.Thefinalconcentrationsmustbe50%(v/v)acetonitrileand5%(v/v)formicacid.CentrifugeTubesThe0.5-or1.5-mLplasticmicrocentrifugetubesthatareusedarepurchasedaslow-binding,siliconizedtubes.Alltubesarerinsed,inUSP-gradeet

7、hanolandair-driedpriortouse.TheDigestionProcedureThevolumesnotedinthisprocedureareforproteinbandscutfrom2Dgels.Thesebands,orspots,areontheorderof2mmto4mmindiameter,withagelthicknessof1mm,andhavetotalvolumeslessthan~20µL.Thevolumesgiveninthisprocedu