ApplicationNotes | Westernblot:Identifies~200kDabandinreducedsamples.UseTrisbasedbuffersforbestresultsasepitopeisphosphatedependent;DifferentregionsoftheCNSweredissectedoutimmediatelyandhomogenizedinBUSTbuffer(8Murea,0.5%SDS,2%13-mercaptoethanol,and50mMTris,pH7.4)ataratioofiml/100mgwettissue.ThetissuesUSPensionsweresonicatedwellandcentrifugedinaTL-100ultracentrifuge(BeckmanInstruments,Fullerton,CA)at100,000g
for1hat25°C,andtheproteinconcentrationsinthesupernatantsweredetermined.Proteinswereseparatedon7.5%SDS-PAGEgels,{Tu.Petal(1995)JCellBiol129:1629-1640}
Immunohistochemistryonfrozensections;noteffectiveonparaffintissuesinoriginalwork.{Cardenetal,1987}.
Optimalworkingdilutionsmustbedeterminedbyenduser. |