ApplicationNotes | Immunohistochemistryonformalinfixedtissue:1:1,000.(RecommendCat.No.AB1576P).ImmunocytochemistryonPC-12cells:1:1,000.(RecommendCat.No.AB1576P).Immunoblotting:1:5,000.CSPsshouldberesolvedon12-15%SDS-PAGEgelsunderNON-REDUCINGconditions.CSPstypicallymigrateas33-35kDaspeciesinvertebratesamples(dimersmayalsobeseen).IfonemustperformSDS-PAGEunderreducingconditions,itisnecessarytoalkylatetheCSPstopreventsmearing.ToensureproperidentificationofCSPs,samplesmaybedeacylatedbytreatmentwith1Mneutralhydroxylamineor0.1MKOHinmethanolresultingina6-7kDashiftinelectrophoreticmobilityoftheCSPband.Deacylationwillusuallyresultimnsomelossofreactivityonwesternblotssosufficientproteinshouldbeloaded.Alternatively,becauseallCSPsappeartobeacidic(approximatepIof4.6-4.8)2-DgelelectrophoresismaybeusefulltodistinguishsomeCSPs.Finally,somesampletreatmentsmaycausepartialdeacylationofCSPsresultinginasmearofimmunoreactivitybelowthemassofthefattyacylatedmonomer.Optimalworkingdilutionsmustbedeterminedbytheenduser. |