PreparationofNuclearExtractsforGelShiftsandWesternsBlots

Thefollowingprotocolisoptimizedforabout10⁷cells(nearconfluent10cmplate).NOTE:ALLCOMPONENTS(BUFFERS,PROTEASEINHIBITORS,ETC)MUSTBEKEPTONICEDURINGTHEENTIREPROCEDURE.

1.WashplateswithPBStwotimes.

2.Prepare5mlofBufferAina15mlconicalc’fugetube,andaddthefollowing:

-5ulofeachproteaseinhibitor(leupeptin,PMSF,aprotinin,pepstatin,andDTT)

-200ulof10%IGEPAL.

Add0.5mlofthispreparationdirectlytoeachplate,andwait10min.atroomtemp.

3.ScrapecellswithNEWsterilescraper,thenpipetupanddownwithP1000severaltimestodisruptcellclumps.NOTE:youwillhavenearly1mloflysateatthispoint.

4.Transferthislysatetoa1.5microcentrifugetube,placeonice.

5.Centrifugeat4Cattopspeed(15000xg)for3min.

6.Placetubesonice.

7.Savesupernatant(cytosolicfraction)forluciferase/ß-galassay,ifdesired.Otherwise,discardthesupernatant.

8.Prepare1mlofBufferBina1.5mlEppendorf,andadd1ulofeachproteaseinhibitorasabove,butthistimeDONOTaddIGEPAL.Add150mlofthisbuffertoeachtube,andresUSPendpelletbypipetingupanddownwithaP200.Placeonice.

9.Shakevigorouslyat4Cfor2h.

10.Centrifugeat4C,topspeedfor5min.MeasureBradford(protein)concentrationusing5ulofeachsample,thenaliquot15ulaliquotsintoprechilled0.5mlprelabeledmicrocentrifugetubes,freezeinliquidnitrogen,andstorein–80Cfreezer.

BufferA:

10mMHEPES,pH7.9

10mMKCl

0.1mMEDTA

Addjustbeforeuse:1mMDTT,0.5mMPMSF,5ulof10ug/ulofaprotinin,leupeptin,andpepstatinAto5mlofbuffer.

BufferB:

20mMHEPES,pH7.9

0.4MNaCl

1mMEDTA

10%Glycerol

Addjustbeforeuse:DTT,PMSF,aprotinin,leupeptin,pepstatinAasabove.