IMMUNOPRECIPITATION
As a starting point, a run-of-the-mill IP will use 100ug protein (range 50-250ug), 2 ug primary
Ab range 1-2 ug), 2 ug secondary Ab (range 0.5-2 ug), and 100 ul protein A-agarose slurry (50 ul
/ ul primary) in a working volume of 0.5 ml.
Bring 100 ug of an SDS-brain homogenate to 500 ul with IP buffer (PBS containing 5 mM
EDTA, 0.5% NP40, 0.5% deoxycholate, 0.2% SDS and 1x protease inhibitor; if the homogenate
was made with sarcosyl, must use 50 mM Tris pH 8.0/ 150 mM NaCl instead of PBS). Make
sure that the volume of homogenate is 10% or less of the final volume.
Add 2 ug primary Ab to the homogenate. Either add the antibody directly to the homogenate/IP
buffer solution or boil the solution for 10 min, centrifuge for 10 min, cool briefly then add the
Ab. The better method has to be determined empirically for each antigen. NOTE: Never boil
samples containing NP40 but not SDS - on its own, boiling causes NP40 to precipitate.
Incubate the brain homogenate with primary antibody overnight at 4 C with gentle agitation (i.e.
Nutator).
Add 50 ul protein A-agarose (Pierce cat # 20333) per ug primary Ab and continue the incubation
at 4 C with gentle agitation for another hour. Note that protein A-agarose is particularly hard to
pipet. Make sure the slurry is mixed by inversion immediately before each aliquot is removed.
Look to see that each agarose pellet looks to be the same size, and add more if needed to bring
all pellets to the same level.
If the primary Ab was a mouse IgG1, include 2 ug rabbit anti mouse secondary antibody to
bridge between the protein A and the primary. Protein A binds antibodies from many species,
including rabbit and goat, but does not bind mouse IgG1 (though it does bind mouse IgG2). If a
bridging Ab is needed, extend the 4 C incubation to 2 hours.
Centrifuge the IP solution for 3 minutes at 3krpm to collect the agarose. Remove the supernatant
- this can be stored at -20 C and re-IP'ed if the protein sample is precious. To the pellet, add 0.5
ml IP buffer, vortex, and wash the protein A-agarose beads for 30 minutes at 4C with gentle
agitation.
Centrifuge 3 minutes at 3krpm. Remove and discard supernatant. Add 0.5 ml IP buffer and
transfer the beads to a clean Eppendorf tube. Wash the old tube with an additional 0.5 ml IP
buffer, and combine this with the transferred solution. Vortex to mix.
Centrifuge 3 minutes at 3krpm. Remove and discard supernatant. Add 0.5 ml IP buffer and
repeat.
Add 30 ul 2X Laemmli sample buffer to the protein A-agarose pellet and boil 5 minutes.
Centrifuge 2 minutes at max speed, then load 20-25 ul supernatant onto acrylamide gel.
NOTES:
IP buffer used for washing the pellet does not need protease inhibitor.
The final pellet can be stored frozen after adding 2x Laemmli buffer. If storing the pellet for
more than a few days, add 2x Laemmli buffer, boil for 5 minutes, centrifuge briefly, and wrap
the tube in Parafilm. Boiling releases the antibody and antigen into solution from the protein A,
and wrapping the tube helps to prevent evaporation of the pellet.
Whether to add b-ME to the 2X Laemmli buffer needs to be determined empirically based on
where the antigen runs relative to the precipitating antibody. After boiling in the presence of bME, the heavy and light chains separate, and run at approximately 50 and 25-30 kD respectively.
Without b-ME, IgG runs at 125-150 kD.