PreparationofBrainCytosol

1.Chillaglassplateonice.

2.Placebraintissueonthechilledglassplateandcutslicesofbrainintosmallpieces.

3.ResUSPendthepiecesinice-coldRIPAbufferwithoutthedetergentsNP-40andNadeoxycholate.Use1Lper250goftissue

4.Performthefollowingstepinacoldroom:Transferthesuspendedtissuetoablenderandhomogenizeonlowpowerwithfour30secondbursts.Letthehomogenatecoolforoneminutebetweenbursts.Repeatburstsuntilthehomogenateissmooth.

5.Transferthehomogenatetopre-chilledcentrifugetubes.

6.Centrifugeat2900xgfor20minutesat4.Thisstepsedimentsunbrokencellsandnuclei.Discardthepellet.

7.Transferthesupernatantfractiontoanothercentrifugetube.

8.Centrifugethissupernatantfractionat29,000xgfor45minutesat4.

9.Transferandsavethesupernatantfraction.Thisfractioncontainsbraincytosolandmicrosomalmembranes.Concentratethebraincytosolproteinsbyprecipitationwithglacialaceticacidasdescribedbelow.

10.Resuspendthepelletin10mlofice-coldRIPAbuffercontainingdetergents(pleaseseeprotocol"PreparationofModifiedRIPABuffer").

11.Centrifugetheresuspendedpelletat15,000xgfor20minutesat4.

12.Transferthesupernatantfractiontoanothertube.Thisfractioncontainssolubilizedmitochondrialandplasmamembraneproteins.

ConcentrationofBrainCytosolicProteinsbyPrecipitationwithGlacialAceticAcid

CAUTION:Glacialaceticacidiscaustic.Performthefollowingstepinahoodandweargloves,goggles,andalabcoat.

1.Acidifythebraincytosolfraction(Step9above)topH4.5byslowlyaddingandmixingdropsofglacialaceticacid.

2.Sedimenttheprecipitatedproteinsbycentrifugingtheacidifiedsampleat15,000xgfor20minutesat4.Discardthesupernatantfraction.

3.Dissolvethepelletin10mlofdesiredbuffer.

4.Determineproteinconcentrationbyastandardmethod.