Email This Protocol    Print This Protocol

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.


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


  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.
Email This Protocol    Print This Protocol
Email This Protocol    Print This Protocol

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).

Solutions and Reagents:
20 mM Tris-HCl, pH 7.5 1 µg/ml aprotinin
150 mM NaCl 1 mM Na3PO4
1% NP-40 1 mM PMSF
2 mM EDTA 5 mM NaF
1 µg/ml leupeptin 3 mM Na4P2O4
Protein A/G sepharose:
Transfer 30 µl protein A or G sepharose per sample in a microcentrifuge tube,
wash twice in cell lysis buffer to remove preservative (add cell lysis buffer up
to 800 µl and spin down in benchtop centrifuge). Resuspend protein A or G
sepharose in cell lysis buffer at 100 µl per sample.
5X SDS sample Buffer:
312.5 mM Tris-HCl (pH 6.8)
10% SDS (w/v)
250 mM DTT
0.05% Bromophenol Blue (w/v)
Use at 1X
10X SDS Running Buffer:
Dissolve 144 g of Glycine, 30 g of Tris base and 10 g SDS in 800 ml of
distilled H2O.
Add distilled H2O to 1 liter
Use at 1X
Transfer Buffer:
2.25 g Tris base
10.5 g Glycine
1 g SDS
200 ml Methanol
Add distilled water to 1.0 L
Sample preparation:
1. Collect cells and transfer to a microcentrifuge tube and centrifuge at 3000
rpm for 5 min at 4°C.
2. Discard the supernatant and store the pellet at -80°C or process immediately
as described below.
1. Remove samples from -80°C freezer and place on ice.
2. Immediately add 800 µl ice-cold lysis buffer to the samples and vortex, then
incubate for 30 min on ice.
3. Spin lysates at 14,000 rpm in a pre-cooled centrifuge for 10 min.
4. Transfer 720 µl supernatant to a fresh pre-cooled microcentrifuge tube containing
washed protein A or G agarose bead slurry (30 µl packed beads) and
rock the mixture for 30 min at 4°C. Collect the agarose beads by pulsing in a
microcentrifuge (5 seconds at 14,000 rpm, 4°C).
5. Remove supernatant and place in a fresh pre-cooled microcentrifuge tube
containing ~4 µg specific antibody and rock gently at 4°C for 2 hrs. Capture
the immune complex by adding 100 µl of washed protein A or G agarose
bead slurry (30 µl packed beads) and rock the mixture for 1 hr at 4°C.
6. Collect the agarose beads by pulsing in a microcentrifuge (5 seconds at
14,000 rpm, 4°C). Aspirate and discard supernatant. Wash the beads 3 times
with ice-cold cell lysis buffer.
7. After final wash, remove supernatant and add 40 µl 2X SDS sample buffer.
Boil for 5 min at 95°C and spin down the beads at maximum speed
in a microcentrifuge for 5 min at room temperature. Carefully pipette off
8. Load up 30 µl of sample in each well of a 1.5 mm thick gel. Run gel according
to manufacturer’s recommendations and continue with immunoblotting
using BioLegend’s Western Blotting protocol, p. 31 (alternately, radiolabeled proteins prepared from target cells can be used to directly visualize the
immunoprecipitated protein).
1. The choice of lysis buffer depends on the location of the protein (membrane,
cytosolic, nuclear).
2. Immunoprecipitates allowed to incubate overnight may have a higher
background than ones processed for shorter periods of time due to timedependent
aggregation or denaturation of cellular proteins.
3. Always use an isotype-matched irrelevant control antibody (monoclonal) or
same-species serum from a non-immunized animal to remove non-specific
antibody binding in cellular lysates.
Email This Protocol    Print This Protocol
Email This Protocol    Print This Protocol
Santa Cruz Biotechnology

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.

Email This Protocol    Print This Protocol
scroll up