General Protocol for Immunoprecipitation
1. Culture cells in 100mm dish, and stimulate cells as designed.
2. Stop stimulation
For adherent cells: Place cells on ice and aspirate the medium. Immediately add 800ul lysis buffer to the
plates,
scrape the cells and transfer to eppendorf tubes.
For suspension cells: Add 800ul ice cooled HBSS and spin cells
briefly at 2500 rpmx3 minutes at 4
centigrade, aspirate the supernatant, and add 600ul lysis buffer to the pellet.
Pipette up and down a few
times to break the pellet.
For control. stimulate
similar to plate with expected maximal signal, but later IP with pre-immune serum.
3. After scraping the cells (or pipetting the pellet)
in lysis buffer, incubate lysate on ice for 30 minutes while
inverting gently 2-3 times.
4. Spin lysate in eppendorf
tubes at 1,2000 rpmx30 minutes at 4 centigrade and transfer the supernatant to a
new tube.
5. For the lysate, add
your IP antibody at a desired concentration.
For the preimmune tube, add 5ul pre-immune serum.
Incubate on ice for 1-2 hours with occasional vertex.
6. Wash beads (Pansorbin) with lysis buffer, then spin at 3,000 rpm for
10 minutes at 4 centigrade. Prepare
50ul for each sample.
7. Incubate samples with 50 ul beads per tube for 1 hour
on ice with gentle inversion every n10 minutes.
8. Spin at 3,000 rpm at 4 centigrade for 5 minutes and aspirate supernatant.
9. Wash
with lysis bufferx3 times with same spins,
10.Aspirate final wash completely.
11.Add 25 ul 2x protein loading buffer and vortex. Incubate
at 100 centigrade for 5 minutes. Spin at 6,000
rpm at 4 centigrade for 5 minutes. Run on SDS-Page gel just
like regular Western blot.
Lysis buffer format:
1% Trixon X-100
5 mM EDTA
50 mM NaCl
50 mM NaF (for phosphotate- antibody)
10mM
Tris-HCl, pH7.6
0.1% BSA
1% Aprotonin
distilled water
The following agents need to be added immediately before use.
2 mM sodium orthovanadate
10
ul Phenylarsine oxide (PAO)