Solution Preparation
RIPA buffer:
50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton
X 100, 1% Na deoxycholate, 0.1% SDS, 1 mM PMSF, 1 µg/mL
aprotinin, 1 µg/mL leupeptin.
PBS, pH7.4:
10 mM Na2HPO4, 1.8 mM KH2PO4, 50 mM NaCl, 2.7 mM KCl
1X Sample buffer:
65 mM Tris-Cl, pH8.0, 10% (v/v) glycerol, 2.3% (w/v) SDS,
0.01% bromophenol blue, 1% DTT.

PROTOCOL
1. Harvest cells with PBS-EDTA or Trypsin, and count
cells.
2. Lyse the cells in prechilled RIPA buffer (1 ml/107 cells)
for 1 hr rocking at 4°C.
3. Centrifuge for 20 min at 14,000 g at 4°C.Transfer super
natant to a new tube.
4. Prepare protein A/G agarose beads by washing twice
with PBS and restoring to a 50% slurry bead suspension
with RIPA buffer.
5. Pre-clear the cell lysate by adding 50 ml of bead
slurry per ml of cell lysate and incubate at 4°C rocking
for 10 min. Centrifuge for 10 min at 10,000 g at 4°C.
Transfer supernatant to a new tube.
6. Prepare IP reaction by pipetting 0.5 ml pre-cleared cell
lysate (corresponding to 5x106 cells) into a new tube.
Add 1-4 mg antibody per reaction and incubate rocking
for 3 hrs on ice. The optimal amount of antibody rquired
to immunoprecipitate the antigen from a given cell ly
sate should be empirically tested.
7. Add 50 ml of 50% slurry beads and rock for 1 hr at 4°C.
8. Centrifuge sample at 10,000 g for 15 sec in
microcentrifuge. Carefully discard the supernatant.
9. Wash beads twice with 1 ml RIPA buffer (to remove
non specifically associated proteins) and then 3 times
with 1 ml PBS to remove detergents.
10.Finally, resuspend beads in 60 ml Sample buffer, and
boil at 95°C for 5 min. Centrifuge sample at 10,000 g
for 15 sec in microcentrifuge before loading on SDSPAGE.

（Abgent提供）