Immunoprecipitation of a-actinin-4 using 985 antibodies (and pre-immune
control):
(1) Prepare cold rinse buffer and IP buffer with protease
inhibitors (ImmunoPrecipitation buffer -- 100 mM NaCl, 10 mM HEPES, pH 7.8,
0.1% TX-100).
(2) Take cells (100mm dish) from the tissue culture incubator.
(3)
Rinse cells by 3x with 10 mM HEPES, pH7.8, 0.02% NaN3.
(4) Extract cells with IP buffer on ice.
Add 3 ml IP buffer to each 100 mm dish of cells. Scrape cell. Transfer to a 50 mL conical tube. Passage cells
through a 25G needle/syringe 10-20X.
(5) Spin cell lysate in eppendorf tubes (1.5 mL) in the cold
room at top speed for 30 min.
(6) Take supernatant to a 15 mL conical tube.
(7)
Add 2X 1.2 mL supernatant to 2 x1.5 mL eppendorf tubes. Save the rest of supernatant in 15 mL conical.
(8)
Add 20 ul 985 to 1 tube; add 20 ul preimmune-985 to 1 tube.
(9) Rotate tubes in cold room
for at least 3 hours.
(10) Wash Protein A-sepharose beads 3X with IP buffer. Add 2X 50 uL
beads to eppendorf, add 1 mL IP buffer. Spin at top speed for 3 minutes. Discard supernatant (gently pipet supernatant
without disturbing beads). Add 1 mL IP buffer. Spin.....repeat 3X.
(11) Spin IPs at top speed in eppendorf for 30
min in the cold room.
(12) Take 1 mL supernatant to 50 uL washed Protein A-sepharose beads.
(13) Rotate
for at lease 3 hours in the cold room.
(14) Wash beads 6X with IP buffer. Be careful not to disturb beads.
(15)
Store IP beads at -20 C until use.
