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Protocol of Immunoprecipitation (IP)

Immunoprecipitation (IP) is a method of purification and enrichment of target proteins depending on antigen-antibody specific reactions. Antibody combines with target proteins in samples and then the antibody can react with protein A/G or sepharose beads coupled with secondary antibody or magnetic beads. There are two major different methods to immunoprecipitate proteins. The first approach (Protocol A) is to mix antibody with protein sample, followed by addition of Protein A/G support, achieving high purity of protein. However, the antibodies are also co-eluted with protein of interest which sometimes creates difficulties in western blot (WB). The alternative (Protocol B) is to bind antibody to the Protein A/G beads and then mix with the antigen, which gives lesser yield than the first approach but avoids the problem of co-elution of antibodies. Refer to our Troubleshooting of Immunoprecipitation (IP) when you get in trouble.

Reagents:

  1. Non-denaturing lysis buffer
    • 20 mM Tris-HCl pH 8
    • 137 mM NaCl
    • 1% Nonidet P-40 (NP-40)
    • 2 mM EDTA.

    Add protease inhibitors before use

  2. Denaturing lysis buffer
    • 1% SDS
    • 5 mM EDTA

    Add 10 mM dithiothreitol or beta-mercaptoethanol and protease inhibitors before use.

  3. Wash buffer
    • 10mM Tris, pH 7.4
    • 1mM EDTA
    • 1mM EGTA, pH 8.0
    • 150mM NaCl
    • 1% Triton X-100
    • 0.2mM sodium orthovanadate

    Add protease inhibitors before use

  4. Glycine elution buffer: 0.2 M glycine, pH 2.6
  5. SDS elution buffer: 2 x SDS loading buffer
  6. Pre-urea wash buffer
    • 50 mM Tris pH 8.5
    • 1 mM EGTA
    • 75 mM KCl
  7. Urea elution buffer:
    • 6-8 M Urea
    • 20 mM Tris pH 7.5
    • 100 mM NaCl

Preparation of Cell Lysates

Lysates from cell culture (non-denaturing)

  1. Place the cell culture dish on ice. Wash the cells with ice-cold PBS.
  2. Drain the PBS. Add ice-cold lysis buffer.

    3. Note: 1 mL/107 cells/100 mm2 dish/150 cm2 flask; 0.5 mL/ 5x106cells/60 mm2 dish/75cm2 flask.

  3. Scrape adherent cells off the dish using a cold plastic cell scraper. Gently transfer the cell suspension into a pre-cooled microcentrifuge tube. Maintain constant agitation for 30 min at 4°C.
  4. Centrifuge in a microcentrifuge at 4°C. Aspirate the supernatant and place in a fresh tube kept on ice. Discard the pellet.

    5. Note: The centrifugation force and time depending on the cell type. A guideline is 20 min at 12,000 rpm.

  5. Proceed with the immunoprecipitation.

Lysates from cell culture (denaturing)

  1. Add 100 μL denaturing lysis buffer to 0.5-2x107 cells.
  2. Mix well by vortexing vigorously for 2-3 s at maximum speed. Transfer the cell suspension to a microcentrifuge tube.
  3. Heat samples to 95°C for 5 min to denature.
  4. Dilute the suspension with 0.9 mL non-denaturing lysis buffer. Mix gently.

    5. Note: The excess 1% Triton X-100 in the non-denaturing lysis buffer quenches the SDS in the original denaturing buffer.

  5. Fragment the DNA by passing the lysed suspension 5-10 times through a needle attached to a 1 mL syringe.

    6. Note: Repeat mechanical disruption until the viscosity is reduced to manageable levels.

  6. Incubate on ice for 5 min.
  7. Proceed with the immunoprecipitation.

Lysates from tissue

  1. For a ~5 mg piece of tissue, add about 300 μL lysis buffer rapidly to the tube and homogenize with an electric homogenizer.
  2. Rinse the blade twice with another 300 μL lysis buffer per rinse. Maintain constant agitation for 2 h at 4°C.

    3. Note: Volumes of lysis buffer must be determined in relation to the amount of tissue present.

  3. Centrifuge for 20 min at 12,000 rpm at 4°C in a microcentrifuge. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice. Discard the pellet.
  4. Proceed with the immunoprecipitation.

Immunoprecipitation

Protocol A: Immunoprecipitation with antibodies in solution

  1. Add 10-500 µg cell lysate plus the recommended amount of antibody into the tube on ice.

    2. Note: These amounts will be chosen depending on the abundance of the protein and the affinity of the antibody for the protein. A recommended guideline: 1-5 µl polyclonal antiserum, 1 µg affinity-purified polyclonal antibody (pAb), 0.2-1 µl ascites fluid (monoclonal antibody, mAb), 20-100 µl culture supernatant (mAb)

  2. Incubate the sample with the antibody for 1-12 h or overnight at 4°C, preferably under agitation.

    3. Note: The incubation time depends on the amount of protein and affinity properties of the antibody.

  3. Meanwhile prepare the sepharose beads. If using a mAb, choose protein G-coupled sepharose beads. If using a pAb, protein A-coupled sepharose beads are usually suitable.

    4. Note: For IgM antibody, do not use protein-A or protein-G conjugated beads. Use Goat anti-Mouse IgM (or polyvalent Ig, or anti heavy chain) beads.

  4. Mix the slurry well. Add 70-100 µl of the beads to each sample. Always keep samples on ice. Beads will tend to stick to the sides of the tip so try to minimize the movement in the pipette and use a tip cut 5 mm from the top.
  5. Incubate the lysate-beads mixture at 4°C under rotary agitation for 4 h. Proceed to wash step.

    6. Note: The optimal incubation time can be determined in a preliminary experiment.

Protocol B: Immunoprecipitation with antibody-agarose conjugate

  1. Add approximately 70-100 µL of slurry of protein A-, or G-, or L-agarose conjugate to microcentrifuge tubes.
  2. Add 10 µL of primary antibody. Incubate the antibody-bead mixture for 1-4 h at 4°C by gently mixing the mixture on a suitable shaker.
  3. Centrifuge at 1,000-3,000 x g for 2 min at 4°C. Discard the supernatant.
  4. Add 1 mL lysis buffer to the mixture by keeping gentle agitation. Centrifuge at 3,000 x g for 2 min at 4°C. Repeat this washing step twice.
  5. After washing the beads and antibody mixture, add 10-50 μg of cell lysate.
  6. Incubate the cell lysate-bead/antibody conjugate mixture at 4°C under rotary agitation for 4 h or overnight.

Washing

  1. When the incubation time is over, centrifuge the tubes. Remove the supernatant by aspiration without disturbing the beads. The protein of interest should now be specifically bound to the antibody coating the beads.
  2. Wash the beads with washing buffer or lysis buffer three times.
  3. Carefully remove the last supernatant. The complex is now ready for elution from beads.

Elution

Three methods can be used to elute the protein from the beads. SDS buffer is the harshest, which will also elute non-covalently bound antibodies and antibody fragments along with the protein of interest. Glycine buffer gently elutes the protein with reduced amount of eluted antibody.

SDS buffer elution

The antigen-antibody complex is eluted from the beads by heating or boiling samples in loading buffer with denaturant SDS. This method is advantageous because the extraction method is highly efficient and the resulting sample is more concentrated.

  1. Elute 50 µL of beads by heating in 50 µL of 2 x SDS loading buffer without DTT for 10 min at 50°C.
  2. Pellet beads. Transfer supernatant to a new tube and add DTT at 100 mM (elution 1).
  3. Add 50 µL 2 x SDS buffer with DTT to pelleted beads (elution 2).
  4. Boil the eluted samples for 5 min. Analyze content of the sample by WB. In general, there should be target protein in both elution 1 and 2 but elution 2 will have more IgG contamination than elution 1.

Glycine buffer elution

The complex is eluted from the beads by acidification using a buffer containing 0.1-0.2 M glycine, pH 2.0-3.0. The low pH of glycine weakens the interaction between the antibody and the beads. This method is advantageous as beads can be reused after removal of the glycine buffer. However, the eluted sample should be immediately neutralized with Tris, pH 8.0-8.5.

  1. Elute 50 µL of beads with 3 x 50 µL 0.2 M glycine pH 2.6 by incubating the sample for 10 minutes with frequent agitation before gentle centrifugation.
  2. Pool the eluate. Neutralize by adding equal volume of Tris pH 8.0.
  3. Neutralize the beads by washing 2 x with 150 µL lysis buffer (without detergent) and pool with eluate.
  4. Run the samples on a WB to check the precipitation of proteins.

Urea buffer elution

This method is advantageous for mass spectrometry (MS) because the sample can be digested by proteolytic enzymes.

  1. Wash beads with pre-urea wash buffer. Remove all residual supernatant.
  2. Add 2-5 volumes urea elution buffer. Rotate for 30 min at RT with frequent agitation before gentle centrifugation.
  3. Repeat this process at least twice more to ensure that the entire captured complex has been released from the beads. Pellet beads and remove urea to a new tube.
  4. Run the samples on a WB to check the precipitation of proteins.
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