Co-immunoprecipitation Protocol Co-IP Protocol

Once samples have been prepared, they can be frozen at -80°C for long-term storage, or be used immediately for immunoprecipitation. Protein concentration should be determined by BCA or Bradford assay. As a guideline, the suggested amount of total protein per IP is 500-1,000 μg at approximately 1 μg/μl.

Cells

1. Pre-cool a refrigerated centrifuge to 4°C.
2. Wash cells 3x with ice-cold PBS, then add ice-cold lysis buffer containing protease/phosphatase inhibitors and incubate on ice for 5 minutes.
1. 1 ml per 10⁷ cells/100 mm² dish/150 cm² flask; 0.5 ml per 5x10⁶ cells/60 mm² dish or 75 cm² flask.
2. Reduce the volume of lysis buffer accordingly if a higher protein concentration is required.
3. Scrape cells off dish using a cold plastic cell scraper then transfer the cell suspension into a pre-cooled microcentrifuge tube.
4. Maintain constant agitation for 30 min at 4°C.
5. Centrifuge at 4°C for 15 minutes at ~16,000 x g.
1. This is a guideline and may require optimization depending on cell type.
6. Gently remove the tubes and place on ice. Transfer the supernatant to a fresh tube on ice. Discard the pellet.

Tissue

1. Dissect the tissue of interest quickly, ideally on ice to reduce protease activity.
2. Wash briefly with ice-cold PBS.
3. Cut the tissue into smaller pieces and place into a microcentrifuge tube. Immerse in liquid nitrogen to snap freeze the tissue.
1. Samples can be stored at this point at -80°C, or they can be immediately homogenized.
4. Add 300 μl non-denaturing lysis buffer per 5 mg tissue. Homogenize with an electric homogenizer for three periods of 10-15 s each.
5. Keep the sample on ice for 30 minutes, vortexing occasionally. This time may need to be extended for some tissues.
6. Centrifuge the lysate at 4°C for 15 minutes at ~16,000 x g, then aspirate the supernatant and transfer to a fresh tube on ice. Discard the pellet.
1. Centrifugation steps are highly dependent on tissue type and the protein target. For example, extra centrifugation steps in specific buffers are necessary to isolate membrane or synaptic proteins.

1. Prepare the beads. When working with beads, it is recommended to use wide-bore pipette tips or tips with the end cut off to prevent damage to the beads.
1. Bead preparation:
1. Vortex the beads for 30 s to resuspend, then move 50 μl to a fresh tube. More beads may be required to precipitate out the larger amount of protein needed for some applications, such as MS.
2. Pellet by magnetic field, remove the supernatant and resuspend in 500 ul lysis buffer.
3. Magnetically separate and remove the supernatant. Repeat twice.
2. Dilute isotype control antibody in PBS-T to 5-25 μg/ml and add 400 μl (2-10 μg antibody) to the prepared beads.
1. These quantities can be increased for larger amounts of beads.
3. Incubate with gentle agitation for 30 minutes at room temperature or 2 hours at 4°C.
4. Perform magnetic separation and discard the supernatant.
5. Wash 3x, discarding the supernatant each time.
6. Add 500 μl lysate to the pelleted beads and gently resuspend.
7. Incubate with gentle agitation for 2 hours at 4°C to pre-clear the lysate.
8. Magnetically separate and move the cleared supernatant to a fresh tube. Discard the bead pellet.

1. Equilibrate fresh magnetic beads as in the pre-clearing step.
2. Antibody addition to beads: Dilute IP antibody in PBS-T to 5-25 μg/ml and add 400 μl (2-10 μg antibody) to the prepared beads.
1. These quantities can be increased for larger amounts of beads.
2. An alternative approach is to add antibody to the lysate first, before adding the beads to capture antibody-antigen complexes. See the full Co-IP Guide for details.
3. It is also possible to covalently bond the antibody to the beads to avoid antibody elution, which is a more time-consuming process.
3. Incubate with gentle agitation for 30 minutes at room temperature or 2 hours at 4°C.
4. Perform magnetic separation and discard the supernatant.
5. Wash 3x, discarding the supernatant each time.
6. Lysate addition to bead-antibody complexes: Add 500 μl lysate to the pelleted beads and gently resuspend.
7. Incubate with gentle agitation for 2 hours at 4°C to allow the antibody-bead complexes to capture the bait protein.
8. Perform magnetic separation and discard the supernatant. The target bait protein and its binding partners should now be complexed with the antibody on the beads.
9. Wash the beads 3–4 times with 500 μl lysis buffer, or TBS or PBS with 0.2% Tween-20, depending on required stringency for the protein complex of interest. Discard the supernatant between each wash.
10. Resuspend in 100 μl PBS and transfer to a fresh tube to avoid co-elution of contaminant proteins bound to the tube wall.

SDS Denaturing elution

1. Pellet the beads by magnetic separation and discard the supernatant.
2. Resuspend in an equal volume of SDS sample buffer to the beads.
3. Vortex, and heat at 90-100°C for 5 minutes.
4. Pellet the beads and transfer the supernatant to a new tube, which will contain the eluted antigen.
5. Perform SDS-PAGE and western blot analysis (see our Western Blot Protocols for full details).

Non-denaturing Buffer elution

1. Pellet the beads and discard the supernatant.
2. Resuspend in an equal volume of non-denaturing buffer (low pH glycine buffer or high pH ammonium hydroxide buffer) to the beads.
3. Incubate for 10 minutes at room temperature with gentle agitation.
4. Pellet the beads and transfer the supernatant to a new tube, which will contain the eluted antigen and interacting proteins.
1. The beads can be reused for later IP experiments by washing them in lysis buffer to remove the glycine.
2. These elution steps can be repeated to ensure that as much protein as possible has been eluted from the beads.
5. If using glycine buffer, neutralize the low pH by adding Tris-HCl. This is not necessary for the ammonium hydroxide buffer.
6. Perform SDS-PAGE and western blot analysis (see our Western Blot Protocols for full details) or mass spec analysis. Other assays are also possible given that many proteins will remain in their native state in this buffer.

Urea Buffer denaturing elution

1. Pellet the beads and discard the supernatant.
2. Wash the beads 3x in pre-urea wash buffer.
3. Resuspend in an equal volume of urea buffer to the beads.
4. Incubate for 30 minutes at room temperature with gentle agitation.
5. Pellet the beads and transfer the supernatant to a new tube, which will contain the eluted antigen and interacting proteins.
1. These elution steps can be repeated to ensure that as much protein as possible has been eluted from the beads.
6. Perform SDS-PAGE and western blot (see our Western Blot Protocols for full details) or mass spec analysis.