Endogenous IP (slug in HMECs)

Supplies needed: tubes for samples and beads, beads, razor blade, RIPA buffer, Ab and control Ab

Prepare lysate for each sample in a new tube:

use (1) 15cm plate – 70% confluent per sample (eg. 2 plates for slug IP and IgG control). I resuspend in ~ 420 μl of RIPA

use all of the lysate, but save ~10 -20 μl for input lane

 

Wash the Beads:

Resuspend the beads (use a pipette tip that is cut at the end)

Remove bead slurry to a new tube – use 60 μl of slurry/sample (+1)

Spin down at 5,000 rpm and aspirate media

Wash 3x with 1 mL of RIPA, vortex, spin down and aspirate after each wash

Resuspend with RIPA to obtain a 50% slurry

Aliquot 60μl of slurry to new tubes – spin down to check there is ~ the same amount of beads in each tube and then resuspend the beads again

 

Preclear the lysate:

Add your protein sample (target 1.5-2 mg in 350 μl) to the beads and rock at 4°C for 1 hour

Spin down, collect the supernatant (~350 μl) and transfer to a new tube

Immunoprecipitation

Add (slug) antibody (1:50 dilution) to the 350 μl of precleared lysate

Add add an equal μg quantity of IgG control Ab to the precleared control lysate

Incubate ON in cold room.