Immunoprecipitation Protocols IP Protocols

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

Non-denaturing lysis

1. Pre-cool a refrigerated centrifuge to 4°C.
2. Wash 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.
1. Optional – Pulse sonicate samples for 10-30 seconds then keep the sample on ice for 15 minutes. This will enhance cell lysis but may also damage proteins, so should be optimized to the cell type and target protein.
5. Centrifuge at 4°C for 15 minutes at ~14,000 x g.
1. This is a guideline and may require optimization depending on cell type.
6. Gently remove the tubes and place on ice. Aspirate the supernatant and transfer to a fresh tube on ice. Discard the pellet.

Denaturing lysis

1. Aspirate the culture medium from the cells, place the dish on ice, and wash twice with cold PBS.
2. Add 100 μl lysis buffer to 0.5 – 2 x 10⁷ cells and vortex vigorously for several seconds. Transfer the suspension to a microcentrifuge tube.
1. The suspension may be viscous at this point due to DNA release.
3. Heat samples at 95°C for 5 min.
4. Dilute the suspension with 0.9 ml non-denaturing lysis buffer. Mix gently.
5. Fragment the DNA by pulse sonicating the samples on ice for several seconds, or by passing the sample several times through a needle attached to a 1 ml syringe.
1. This will reduce the viscosity of the sample to manageable levels, otherwise separation of the pellet from supernatant can be affected.
6. Incubate on ice for 5 minutes, then centrifuge the lysate at 4°C for 15 minutes at ~14,000 x g. Transfer the supernatant to a new tube on ice and discard the pellet.
1. The time and speed may vary depending on the cell type.

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 denaturing or non-denaturing lysis buffer per 5 mg tissue. Homogenize thoroughly with an electric homogenizer.
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 ~14,000 x g, then aspirate the supernatant and transfer to a fresh tube on ice. Discard the pellet.

Pre-clearing

1. Add 25 μl of off-target antibody (of the same isotype as the IP antibody) to 500 μl lysate.
2. Incubate for 1 h on ice.
3. While incubating, prepare the beads. When working with beads, it is recommended to use pipette tips with the end cut off to prevent damage to the beads.
1. Bead preparation:
1. If the agarose beads come as a powder, incubate 50 mg of beads in 500 μl PBS for 1 h so they swell up, then centrifuge and discard the supernatant.
2. To equilibrate the beads, add 500 μl PBS containing 0.1% BSA, mix for 1 h with agitation and rinse twice in PBS. Remove the supernatant and add 200 μl of lysis buffer with protease inhibitors.
3. This working slurry can be stored at 4°C for a few days; for longer periods keep the beads in PBS with 0.02% azide but they must be washed thoroughly before use and made up in fresh lysis buffer.
4. Add 50 μl of Protein A or G agarose beads to the lysate.
5. Incubate for 10–30 min at 4°C with gentle agitation.
6. Centrifuge at 14,000 x g at 4°C for 10 min.
7. Aspirate the supernatant and transfer to a fresh tube on ice. Discard the bead pellet.

Immunoprecipitation

8. Antibody addition to lysate: Add 1-10 μg IP antibody or isotype control antibody to 500 μl pre-cleared lysate.
1. Antibody amount should be titrated. Avoid using excess antibody for the amount of target protein, because any unbound antibody will bind to the beads faster than the antibody-antigen complex will. This will occupy the beads’ binding capacity with antibody that is not bound to the target protein, reducing yield.
9. Gently rock the mixture at 4°C for 1-16 h (overnight).
10. Bead addition: Add 50 μl Protein A or G agarose beads to capture the immunocomplex. Gently rock the mixture at 4°C for 1-4 h.
1. Beads should be equilibrated prior to addition, as in the pre-clearing step
11. Centrifuge the mixture at 500–1000 rpm for 30 seconds at 4°C, and discard the supernatant.
12. 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 of interest. Discard the supernatant between each wash.
13. Resuspend in 100 μl PBS and transfer to a fresh tube to avoid co-elution of contaminant proteins bound to the tube wall.

Pre-clearing

1. Prepare the beads. When working with beads, it is recommended to use pipette 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.
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.
3. Incubate with 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 agitation for 30 minutes at room temperature or 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.

Immunoprecipitation

9. Equilibrate fresh magnetic beads as in the pre-clearing step.
10. 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.
11. Incubate with agitation for 30 minutes at room temperature or 2 hours at 4°C.
12. Perform magnetic separation and discard the supernatant.
13. Wash 3x, discarding the supernatant each time.
14. Lysate addition to bead-antibody complexes: Add 500 μl lysate to the pelleted beads and gently resuspend.
15. Incubate with agitation for 30 minutes at room temperature or 2 hours at 4°C to allow the antibody-bead complexes to capture the antigen.
16. Perform magnetic separation and discard the supernatant. The target protein should now be complexed with the antibody on the beads.
17. 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 of interest. Discard the supernatant between each wash.
18. 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 centrifugation or 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 by centrifugation (10,000 rpm for 2 minutes) or magnetic separation 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).

Glycine Buffer elution

1. Pellet the beads by centrifugation or magnetic separation and discard the supernatant.
2. Resuspend in an equal volume of glycine buffer to the beads.
3. Incubate for 10 minutes at room temperature with gentle agitation.
4. Pellet the beads by centrifugation (10,000 rpm for 2 minutes) or magnetic separation and transfer the supernatant to a new tube, which will contain the eluted antigen.
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. Neutralize the low pH by adding Tris-HCl.
6. Perform SDS-PAGE and western blot analysis (see our Western Blot Protocols for full details). 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 by centrifugation or magnetic separation 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 by centrifugation (10,000 rpm for 2 minutes) or magnetic separation and transfer the supernatant to a new tube, which will contain the eluted antigen.
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.