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Immunoprecipitation

IP

Immunoprecipitation (IP) is most commonly used for the purification and detection of antigens. It relies on antibodies and their affinity to specific antigens, thus allowing the isolation of single proteins from samples.

There are two main methods of immunoprecipitation: (i) Method A: the antibody is first mixed with the sample before being fixed onto an agarose or magnetic bead support, and (ii) Method B: the antibody is first conjugated to a support of agarose or magnetic beads before the addition of the sample. The IP procedure can also be modified such that antigen is bound to the support, allowing the collection and analysis of target antibodies. Bound antigen-antibody complexes are pulled out of the sample via the beads, isolating the protein of interest for analysis through SDS-PAGE and Western Blot (WB), which can be used to separate and visualise the protein.

See below for a general protocol for Immunoprecipitation.

A. Reagents and Preparation

Reagents

  • PBS
  • Protein A or G agarose beads
  • Protease Inhibitor: PMSF, or aprotinin
  • Primary Antibody

Lysate Preparation (for cell culture)

Non-Denaturing Denaturing
  1. Wash cells with ice-cold PBS and then drain off PBS.
  2. Add ice-cold non-denaturing cell lysis buffer to the cells (1ml per 107 cells).
  3. Scrape adherent cells off using a pre-cooled plastic cell scraper, then transfer to microcentrifuge tubes and keep them on ice.
  4. Maintain constant agitation of the suspension, using and rocker or an orbital shaker, at 4°C for 15 to 30 mins to lyse cells.
  5. Centrifuge the lysate in a precooled microcentrifuge.
  6. Aspirate the supernatant and transfer to a clean tube and keep on ice, discard of the pellet.
  1. Add denaturing lysis buffer to cells.
  2. Mix thoroughly by vortexing for 3 seconds at maximum speed, and then transfer the cell suspension to a microcentrifuge tube.
  3. Heat samples for 5 mins at 95°C to allow denaturing, and the dilute with non-denaturing lysis buffer.
  4. Note: The solution can be viscous due to the release of DNA. Fragment the DNA by passing the lysed sample through a needle attached to
    a 1 ml syringe, and repeat this mechanical disruption until the viscosity is reduced.
  5. Incubate for 5 mins on ice.

Pre-Clearing Cell Lysate (optional)

  1. Add 100 μl of protein A or G agarose bead slurry per 1 ml of cell lysate.
  2. Incubate at 4°C for 10-30 mins.
  3. Microcentrifuge for 10 mins at 4°C.
  4. Keep supernatant and discard bead pellet.

B. Protocol

Immunoprecipitation

Method A: Immunoprecipitation with antibodies in solution. The antibody is first mixed with the sample before the addition of protein A or G agarose B support.

  1. Add primary antibody to cell lysate, and incubate overnight at 4°C with gentle shaking. Note: Refer to the antibody datasheet for the recommended dilution.
  2. Add 100 μl of protein A or G agarose bead slurry per 1 ml of cell lysate, and incubate at 4°C with gentle shaking for 1-3 hrs.
  3. Centrifuge at 4°C for 30 seconds to collect the immunoprecipitant, discard the supernatant, and wash beads 5 times with cell lysis buffer to remove non-specific binding.

Method B: Immunoprecipitation with agarose-conjugated antibody. The antibody is first mixed with Protein A/G agarose beads, to allow conjugation, before the addition of the sample.

  1. Add 100 μl of protein A or G agarose bead slurry to microcentrifuge tubes.
  2. Add primary antibody, and incubate at 4°C with gentle shaking for 1-4 hrs. Note: Refer to the antibody datasheet for the recommended dilution.
  3. Centrifuge at 4°C for 2 mins, and discard the supernatant. Wash with lysis buffer, twice, with gentle shaking followed by centrifugation.
  4. Add cell lysate, and incubate at 4°C overnight. Note: The optimum incubation time should be determined by user, via preliminary experiments.

Note: If using an IgM antibody, do not use protein A or G conjugated beads, use anti-IgM coupled protein A or G beads.

Elution

Glycine Buffer Elution SDS Buffer Solution
  1. Elute the beads with glycine buffer (0.1-0.2 M glycine, pH 2.0-3.0), 3 times, and incubate the sample for 10 mins with gentle shaking, and then centrifuge briefly.
  2. Pool the eluted sample, and immediately neutralise by adding equal volume of Tris pH 8.0.
  3. Note: Following the removal of glycine buffer, beads can be reused.
  1. Elute the beads by heating 2 x SDS loading buffer, without DTT, for 10 mins at 50°C.
  2. Elution 1: Pellet beads, transfer the collected supernatants to a fresh tube, add DTT at 100 mM.
  3. Elution 2: Add 2x SDS Buffer (with DTT) to pelleted beads.
  4. Boil eluted samples for 5 mins. Note: Although the target protein should be present in both elution 1 and 2, the quantity will be variable and greater IgG contamination in elution 2 is expected.

For more information, try our Immunoprecipitation (IP) Troubleshooting Guide.