As the name suggests, co-immunoprecipitation employs specific antibodies to sequester a protein of interest together with its interacting partners from a solution.
The success of the co-immunoprecipitation procedure depends on
- How specifically the antibody recognizes the protein of interest and
- How well the protein-protein interactions are preserved in a solution. Weakly interacting proteins may lose contact and have difficulties to be co-precipitated with their interacting proteins.
The procedure of co-immunoprecipitation includes the following steps:
Step 1: Cell lysate preparation
The lysate preparation is the first step in Co-Immunoprecipitation. For lysis, tissue culture cells are incubated with lysis buffer on ice for 20 – 30 min. Tissues need to be homogenized in lysis buffer on ice.
There is no universal lysis buffer that can extract all proteins from cells and tissues especially membrane and cytoskeleton-associated proteins without any degradation and denaturation while retaining protein interactions intact. Therefore, one can try different lysis buffers to co-immunoprecipitate specific proteins and their interacting partners.
Step 2: Clearing of cell lysate
Once the lysis is complete, the insoluble fraction of lysate must be removed. Centrifugation at high speed is the most commonly used method to remove insoluble components of cell lysate.
Step 3: Incubation of cleared cell lysate with an antibody specific to the protein of interest
The cleared lysate is incubated with an antibody specific to the protein of interest at 4°C for 4 h to overnight with gentle shaking. The amount of lysate and the antibody depends on the expression of the protein of interest and its interacting partners. Usually, 1 – 5 µg of antibody in 0.4 – 0.5 ml cleared lysate (total protein concentration 0.4 -1µg/µl) is added in cells expressing a moderate to a good amount of protein of interest.
Step 4: Sequestration of Antibody-protein complex by Protein A/G coated agarose beads/magnetic beads
Since antibodies have an affinity towards Protein A/G, agarose beads/magnetic beads coated with Protein A/G are added to the lysate. Antibody together with its protein of interest and associated complex binds to the Protein A/G coated agarose beads/magnetic beads. Generally, 30 min – 60 min incubation at 4°C with gentle shaking is sufficient to sequester all antibody complexes.
Step 5: Washing beads with washing buffer
Beads-bound protein complexes are washed to remove unspecific proteins. The washing buffer can be the cell lysis buffer, PBS, or buffer containing salt solution. Agarose beads need to be centrifuged in order to collect them at the bottom of the microcentrifuge tube while magnetic beads are collected using a magnetic strand.
Step 5: Processing of immunocomplexes for downstream applications
Once the beads-bind immunocomplex is purified it can be further processed depending on experimental plans. Proteins can be eluded from the beads or directly dissociated in SDS-PAGE Laemmli Sample Buffer for analysis by SDS-PAGE.