Coimmunoprecipitation
Summary
Coimmunoprecipitation is used to determine if two proteins bind to one another in a sample.
Also known as:
Co-IP, pull-down
Samples needed
Usually co-IPs are used to determine if two proteins bind to one another in cells, so the cells of interest are needed.
Controls
Researchers should always show whole lysate control, or a sample that has not been subjected to immunoprecipitation. This shows the reader how much of the protein of interest is present in the sample to begin with. Also, researchers should show a control where the pull-down is performed with an antibody that does not bind to the protein of interest.
Method
Coimmunoprecipitation allows a researcher to determine whether two proteins of interest (A and B) bind to one another in a cell lysate or other sample. First, an antibody that is specific to protein A is attached to beads that can be separated from a solution by centrifugation, magnet, or some other method. Then cells of interest are lysed to release the proteins. The lysate is incubated with the anti-A beads as well as control beads that have been attached to an antibody that does not bind protein A. During the incubation, the anti-A antibody binds protein A. Importantly, any other proteins that are also bound to protein A will also be bound to the beads through protein A. The exact buffer used for this incubation and the subsequent washes is important, because the buffer composition will determine what protein-protein interactions are maintained or broken.
Then the beads are washed to remove any proteins that are binding non-specifically, and finally the proteins still bound to the beads are eluted. This leaves the researcher with a solution of protein A and any other proteins that are bound to protein A. Since the question is whether protein A binds protein B, the sample is then subjected to Western blot to detect protein B.
Interpretation
Figure 1. A Western blot showing the results of a coimmunoprecipitation-esque experiment. Relevant section of caption for published figure reads: “C, cell lysates were prepared from IGROV-1… cells and incubated with 1 μmol/L biotinylated-birinapant (B-B), or no compound (Ctr) for 16 hours. Magnetic streptavidin conjugated beads were then added and incubated for an additional 2 hours. Beads were collected by magnetic separation. Following washes, beads were boiled in SDS-PAGE sample loading buffer to elute bound proteins. Western blot analysis was performed to determine IAP association with birinapant.” “Figure 1” by Christopher A. Benetatos et al.[1]. [Image description]
Strictly speaking, one could argue that this is not an immunoprecipitation experiment, because there is no antibody involved. However, the protocol and reasoning are similar, and it is a form of pull-down assay. In this case, the researchers are asking whether the proteins cIAP1, cIAP2, and XIAP bind to the molecule birinapant. A molecule called biotin has been attached to the birinapant. Biotin b