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Co Immunoprecipitation Protocol

Co-Immunoprecipitation (Co-IP) is a widely used technique to isolate a target protein along with its interacting partners from complex biological samples. The following protocols provide a complete overview of Co-IP, from sample preparation to elution. Exact incubation times and volumes may vary depending on the protein, antibody, and beads used, so additional optimization may be required.

Sample Preparation and Lysis

Prepared samples can be stored at -80°C for long-term storage or used immediately for immunoprecipitation. Protein concentration should be determined using a BCA or Bradford assay. Typically, 500–1,000 μg of total protein at approximately 1 μg/μl is recommended per IP.

For Cells:

  1. Pre-cool a refrigerated centrifuge to 4°C.
  2. Wash cells three times with ice-cold PBS, then add ice-cold lysis buffer containing protease and phosphatase inhibitors. Incubate on ice for 5 minutes.
    • 1 ml per 10⁷ cells (100 mm² dish / 150 cm² flask)
    • 0.5 ml per 5×10⁶ cells (60 mm² dish / 75 cm² flask)
  3. Scrape cells using a cold plastic scraper and transfer the suspension to a pre-cooled microcentrifuge tube.
  4. Agitate on ice for 30 minutes, then centrifuge at 16,000 × g for 15 minutes at 4°C.
  5. Transfer the supernatant to a fresh tube on ice and discard the pellet.

For Tissue:

  1. Dissect tissue quickly, ideally on ice, and wash briefly with ice-cold PBS.
  2. Cut tissue into small pieces and snap freeze in liquid nitrogen. Samples can be stored at -80°C or homogenized immediately.
  3. Add 300 μl non-denaturing lysis buffer per 5 mg tissue and homogenize using an electric homogenizer (3 × 10–15 s).
  4. Incubate on ice for 30 minutes, vortexing occasionally.
  5. Centrifuge at 16,000 × g for 15 minutes at 4°C and transfer the supernatant to a fresh tube. Discard the pellet.

Note: Centrifugation steps may need adjustment depending on tissue type or target protein. Additional steps may be required for membrane or synaptic proteins.

Pre-Clearing

  1. Prepare beads by resuspending them and transferring an appropriate volume to a fresh tube. Adjust bead amount based on protein quantity.
  2. Pellet beads using a magnetic field and wash twice with lysis buffer.
  3. Dilute a control antibody in PBS-T (5–25 μg/ml) and incubate with beads for 30 minutes at room temperature or 2 hours at 4°C with gentle agitation.
  4. Wash beads three times with lysis buffer, discarding supernatant each time.
  5. Add 500 μl of the prepared lysate to the beads, resuspend gently, and incubate for 2 hours at 4°C.
  6. Magnetically separate beads and transfer the cleared lysate to a fresh tube.

Co-Immunoprecipitation

  1. Equilibrate fresh beads as in the pre-clearing step.
  2. Add the target antibody to beads (5–25 μg/ml, 2–10 μg antibody) and incubate with gentle agitation for 30 minutes at room temperature or 2 hours at 4°C.
  3. Wash beads three times with lysis buffer.
  4. Add 500 μl lysate to the antibody-bound beads and incubate for 2 hours at 4°C to capture the bait protein and its interacting partners.
  5. Wash beads 3–4 times using lysis buffer or PBS/TBS with 0.2% Tween-20, depending on the desired stringency.
  6. Transfer beads to a fresh tube for elution.

Elution Methods

SDS Denaturing Elution:

  • Resuspend beads in SDS sample buffer, heat at 90–100°C for 5 minutes, pellet beads, and transfer supernatant.

Non-Denaturing Buffer Elution:

  • Resuspend beads in low-pH glycine buffer (0.1–0.2 M, pH 2–3) or high-pH ammonium hydroxide buffer (0.5 M, pH 11) for 10 minutes at room temperature.
  • Pellet beads, transfer supernatant, and neutralize if using glycine buffer.

Urea Denaturing Elution:

  • Wash beads in pre-urea buffer, resuspend in 6–8 M urea buffer, incubate 30 minutes at room temperature, pellet beads, and transfer supernatant.

Eluted samples can be analyzed via SDS-PAGE, western blot, or mass spectrometry. Non-denaturing elution maintains protein complexes for downstream functional assays.

Buffers and Reagents

Denaturing Lysis Buffer:

  • 50 mM Tris, pH 8.0
  • 150 mM NaCl
  • 1% NP-40 or Triton X-100
  • 0.5% sodium deoxycholate
  • 0.1% SDS
  • Add protease and phosphatase inhibitors before use

Non-Denaturing Lysis Buffer:

  • 1% Triton X-100
  • 0.5% NP-40
  • 150 mM NaCl
  • 10 mM Tris-HCl, pH 7.4
  • 1 mM EDTA
  • Add inhibitors before use

Detergent-Free Lysis Buffer:

  • 1x PBS
  • 5 mM EDTA
  • 0.02% sodium azide
  • Add inhibitors before use

Wash Buffer:

  • PBS or TBS with 0.2% Tween-20

Elution Buffers:

  • Glycine buffer: 0.1–0.2 M, pH 2–3
  • Ammonium hydroxide buffer: 0.5 M, pH 11, 0.5 mM EDTA
  • Urea buffer: 6–8 M urea, 100 mM NaCl, 20 mM Tris, pH 7.5