Immunoprecipitation

Immunoprecipitation (IP)

Most often used for the purification and detection of antigens. Immunoprecipitation  utilises antibodies and their affinity to specific antigens to isolate single proteins from a sample.

An antibody for a specific antigen is chosen and bound to a support such as agarose or magnetic beads. Samples are then added and incubated, enabling the target antigen to bind to the immobilised antibody if present. The bound antigen antibody complexes formed can be collected and analysed from this. The procedure may also be modified with antigen bound to the support and target antibodies collected from the sample. The antigen/ antibody complex formed may then be pulled out of the sample using agarose or magnetic beads, isolating the protein of interest from the rest of the sample. SDS-PAGE and WB may then be used to separate and visualise the protein.

Before carrying out IP the cell samples must first be lysed. The best lysis buffer will degrade the cell but leave the protein of interest in its native confirmation, weak non ionic detergents such as NP-40 and Triton X-100 are recognised to minimise degradation, whilst SDS and Sodium deoxycholate are considered stronger ionic detergents increasing degradation.  To optimise the degradation of cell samples detergents should be tested and analysed beforehand. 

Reagents

  • PBS
  • Non-denaturing cell lysis buffer - 20mM Tris, 150mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 Sodium phosphate.
  • Protein A or G agarose beads.

Preparing cell lysate

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 4oC 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.

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 4oC for 10-30 minutes.
3) Microcentrifuge for 10 min at 4oC.
4) Keep supernatant and discard bead pellet.

Immunoprecipitation

1) Add primary antibody to cell lysate and incubate overnight at 4oC with gentle rocking.
2) Add 100 μl of protein A or G agarose bead slurry per 1 ml of cell lysate and incubate at 4oC with gentle rocking for 1-3 hours.
3) Centrifuge at 4oC for 30 seconds, discard the supernatant and wash beads 5 times with cell lysis buffer.