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Immunoprecipitation

  1.  Solubilize the sample in an appropriate detergent (see below) containing protease inhibitors – 1 ml per 6 cm dish for 30 minutes at 4°C then scrape into a microfuge tube (use 2 ml for P100). Spin sample at high speed in microfuge for 15 minutes at 4°C.
  2. Transfer supernatant to a fresh tube containing 2-5 µl of primary antibody. Incubate 1h to overnight at 4°C with mixing. Typically, I give the antibodies about 30 minutes and then add the IP beads. You can also add the antibody and IP beads at the same time.  20-40 µl of a 50% bead slurry, pre-washed in buffer to give 10-20 µl of packed beads. (TIP: cut end off of a 200 ul tip to increase the tip’s open diameter or use large orifice tips.)
  3. Spin down beads in microfuge and wash 3 X 1 ml with cold detergent. Keep samples on ice as much as possible. Remove as much supernatant as possible after final spin. A gel loading tip or 27G needle helps here.
  4. Add 2X or 4X sample buffer, minus reducing agents, to beads and incubate at room temperature for 30 minutes with vortexing. Using reducing agents at this step will give antibody chains in the eluted sample. Use same volume SB as bead volume and remember there will be some dead volume of wash buffer (usually about 50% of bead volume) that must be considered if you plan to load the entire sample onto the gel.
  5. Using an 18G needle, make a hole in the top of the sample tube. Next make a small hole in the bottom of the tube by inserting the needle about half way up the beveled edge…just enough for the tip to pierce the inside. Place the punctured tube into a fresh microfuge tube and place the assembly into a 50 ml conical tube.  Spin in clinical centrifuge 15 seconds at 500 xg.  If this is done right, the sample will spin into the new tube and the beads will be left behind. If needed, add reducing agents to sample and heat 15 minutes at 65 °C.
  6. Run on a gel.

IP Detergents and buffers:

TBS

50mM Tris pH 7.4

10mM Na MoO4

150mM NaCl

 

RIPA:

1.0% Deoxycolate

1.0% Triton X 100

0.1% SDS

In TBS

Note: RIPA is a standard IP detergent, but is harsh and can dissociate protein-protein interactions as well as dissolve the nuclear membrane and release DNA which will need to be removed either enzymatically or by physical shearing.

 

0.09% – 1% NP40:

0.09% -1% NP40 in TBS

 

Note: Low concentrations of NP40 will solubilize the plasma membrane well and maintain protein-protein interactions without solubilizing the nuclear membrane.

 

1% DMNG

1% DMNG in TBS

 

1000X Protease Inhibitor Cocktail:

Leupeptin (Sigma L2884):    1mg/ml

Aprotinin (Sigma A1153):    2mg/ml

E64 (Sigma E3132):        3.57mg/ml

Benzamidine (Sigma B6506):    156.6mg/ml

Pefablock (Roche 1429876):    2M