SolutionPreparation
RIPAbuffer:
50mMTris-HClpH7.4,150mMNaCl,1mMEDTA,1%Triton
X100,1%Nadeoxycholate,0.1%SDS,1mMPMSF,1µg/mL
aprotinin,1µg/mLleupeptin.
PBS,pH7.4:
10mMNa2HPO4,1.8mMKH2PO4,50mMNaCl,2.7mMKCl
1XSamplebuffer:
65mMTris-Cl,pH8.0,10%(v/v)glycerol,2.3%(w/v)SDS,
0.01%bromophenolblue,1%DTT.

PROTOCOL
1.HarvestcellswithPBS-EDTAorTrypsin,andcount
cells.
2.LysethecellsinprechilledRIPAbuffer(1ml/107cells)
for1hrrockingat4°C.
3.Centrifugefor20minat14,000gat4°C.Transfersuper
natanttoanewtube.
4.PrepareproteinA/Gagarosebeadsbywashingtwice
withPBSandrestoringtoa50%slurrybeadsUSPension
withRIPAbuffer.
5.Pre-clearthecelllysatebyadding50mlofbead
slurrypermlofcelllysateandincubateat4°Crocking
for10min.Centrifugefor10minat10,000gat4°C.
Transfersupernatanttoanewtube.
6.PrepareIPreactionbypipetting0.5mlpre-clearedcell
lysate(correspondingto5x106cells)intoanewtube.
Add1-4mgantibodyperreactionandincubaterocking
for3hrsonice.Theoptimalamountofantibodyrquired
toimmunoprecipitatetheantigenfromagivencellly
sateshouldbeempiricallytested.
7.Add50mlof50%slurrybeadsandrockfor1hrat4°C.
8.Centrifugesampleat10,000gfor15secin
microcentrifuge.Carefullydiscardthesupernatant.
9.Washbeadstwicewith1mlRIPAbuffer(toremove
nonspecificallyassociatedproteins)andthen3times
with1mlPBStoremovedetergents.
10.Finally,resuspendbeadsin60mlSamplebuffer,and
boilat95°Cfor5min.Centrifugesampleat10,000g
for15secinmicrocentrifugebeforeloADIngonSDSPAGE.