Immunoprecipitation

This protocol provides a general method suitable for the immunoprecipitation of radiolabeled antigens. Please note that the choice of radio-labeling procedure may be important in the design of an immunoprecipitation experiment. For the labeling of cell surface antigens, lactoperoxidase catalyzed iodination with 125l is recommended, while for intracellular antigens in cultured cells, 35S-methionine incorporation would be more suitable (see Practical Immunology, Hudson, C. and Hay, F.C. Blackwell Scientific Publications).

We recommend that suitable controls are used throughout. Appropriate safety precautions should be taken when working with any radioactive isotopes.

Reagents

1. Solubilizing solution
To 100 ml of Phosphate Buffered Saline (PBS) add:

EDTA, 37.0 mg
EGTA, 38.0 mg
Iodoacetamide, 93.0 mg
Phenylmethylsulfonylfluoride, 17.4 mg
Soybean trypsin inhibitor, 2.0 mg
Aprotinin (20 KIU/ml), 100 μl

Method

1. If using radio-labeled cells, lyse 2 x 107 cells in 0.5 ml of cold solubilizing solution. Incubate for 20 minutes on ice.
2. Centrifuge at 1000 g for 10 minutes to remove insoluble debris.
3. Add lysate to 200 μl of Protein G-Sepharose beads, and incubate overnight at 4°C with tumbling. This precautionary step will remove any material from the lysate that may bind non-specifically to the beads.
4. At the same time in a separate tube, add 200 μl of the monoclonal antibody of interest (at approximately 10-50 μg/ml) to 100 μl of Protein-G Sepharose beads. Incubate with tumbling overnight at 4°C.
5. Remove beads from cell lysate suspension by centrifugation (11,000 g for 5 minutes).
6. Wash monoclonal antibody-loaded beads twice in PBS. Discard the supernatant following centrifugation.
7. Add the cell lysate to the monoclonal antibody-loaded beads and incubate at 4°C for at least 90 minutes with tumbling.
8. Wash the beads 3 times with cold solubilizing solution, harvesting them each time by centrifugation.
9. Analyze immunoprecipitate by eluting beads with a small volume of SDS-PAGE loading buffer. Run the eluate on a suitable SDS-PAGE gel. The gel should be dried and analyzed by autoradiography following a suitable exposure period.

Application Notes:

Immunoprecipitation is a procedure by which proteins or peptides that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics. Immunoprecipitation can also be used to “enrich” a protein population prior to Western blotting. For example, immunoprecipitation with a pan-specific antibody against a protein of interest followed by Western blotting with a modification-specific antibody (such as a phospho-specific antibody or an acetylation-specific antibody).

NOTE: This procedure may be used for cells labeled with radioactive compounds such as amino acids or orthophosphate. (Radioisotope use and disposal should conform to institutional and governmental regulations.) Cell labeling should be carried out in medium lacking the relevant nonradioactive compound. Starving cells appropriately prior to labeling is recommended. Incubate cultured cells (80–90% confluent monolayer in 100 mm cell culture plate or approximately 2–5 x 107 suspension cells in flask).

Example: Following starvation, remove culture medium and replace with methionine-free medium containing 5% dialyzed fetal calf serum and 100 µCi/ml 35S-methionine. Incubate 1 hour at 37º C. For some proteins a longer labeling period (up to 18 hours) is preferable. Wash cells with PBS (Buffers and General Solutions) as necessary to remove unincorporated 35S-methionine.

• Add 1–3 ml ice cold RIPA buffer (sc-24948) to subconfluent cell monolayer and incubate at 4º C for  10 minutes. For suspension cells, add the RIPA buffer to washed cell pellet in a microcentrifuge tube.

• Disrupt cells by repeated passage through a 21-gauge needle or sonication. Transfer to a microcentrifuge tube.

• Optional: Wash cell culture plate with additional 1.0 ml ice cold RIPA buffer and combine with original extract.

• Pellet cellular debris by centrifugation at 10,000xg for 10 minutes at 4º C. Transfer supernatant to a fresh microcentrifuge tube on ice.

• Preclear lysate by adding 1.0 µg of the appropriate control IgG (corresponding to the host species of the primary antibody), together with 20 µl of appropriate suspended (25% v/v) agarose conjugate (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose). Incubate at 4º C for 30 minutes.

• Centrifugation at 3,000 rpm (e.g. Eppendorf 5415D; approximately 1,000xg) for 30 seconds at 4º C.

• Transfer the supernatant, or approximately 100–1000 µg total cellular protein, to a microcentrifuge tube. Add 1–10 µl (i.e., 0.2–2 µg) primary antibody (optimal antibody concentration should be determined by titration) and incubate for 1–2 hours at 4º C. Alternatively, if antibody agarose conjugate is employed, add 20 µl (i.e., 5 µl packed beads) and incubate at 4º C for 1 hour to overnight with mixing; skip the next step.

• Add 20 µl of the appropriate agarose conjugate suspension (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose). Cap tubes and incubate at 4º C on a rocker platform or rotating device for 1 hour to overnight.

• Collect immunoprecipitates by centrifugation at 3,000 rpm (approximately 1,000xg) for 30 seconds at 4º C. Carefully aspirate and discard supernatant.

• Gently wash pellet 2–4 times with 1.0 ml RIPA buffer (more stringent) or PBS (less stringent), each time repeating centrifugation step above.

• After final wash, carefully aspirate and discard supernatant and resuspend pellet in 40 µl of 2x electrophoresis sample buffer (sc-24945).

• Boil samples for 2–3 minutes and subject to electrophoresis and autoradiography. Unused samples may be stored at -20º C.

Optional: After boiling, samples may be centrifuged to pellet the agarose beads followed by SDS-PAGE analysis of the supernatant.