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
- Protein A or G agarose beads
- Lysis Buffer
- Non-Denaturing Lysis Buffer: 20mM Tris, 150mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 Sodium phosphate
- Denaturing Lysis Buffer: 1% SDS, 5 mM EDTA and (added immediately before use) Protease inhibitors15 U/mL DNase1, 10 mM dithiothreitol or beta-mercaptoethanol
- Protease Inhibitor: PMSF, or aprotinin
- Primary Antibody
Lysate Preparation (for cell culture)
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.
Pre-Clearing Cell Lysate (optional)
- Add 100 μl of protein A or G agarose bead slurry per 1 ml of cell lysate.
- Incubate at 4°C for 10-30 mins.
- Microcentrifuge for 10 mins at 4°C.
- Keep supernatant and discard bead pellet.
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.
- 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.
- 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.
- 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.
- Add 100 μl of protein A or G agarose bead slurry to microcentrifuge tubes.
- 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.
- Centrifuge at 4°C for 2 mins, and discard the supernatant. Wash with lysis buffer, twice, with gentle shaking followed by centrifugation.
- 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.
|Glycine Buffer Elution||SDS Buffer Solution|
Note: Following the removal of glycine buffer, beads can be reused.
For more information, try our Immunoprecipitation (IP) Troubleshooting Guide.