Coimmunoprecipitation coIP is commonly used in biomedical re

Co-immunoprecipitation (co-IP) is commonly used in biomedical research to examine whether proteins interact physically with each other. You perform a co-IP procedure to determine if a protein called Elm interacts with proteins called Oak and Red. Because you do not have antibodies against Oak and Red, you genetically engineer cells in which both of these proteins are tagged via the insertion of a short peptide sequence called HA, for which you purchase specific antibodies. First, you permanently link an Elm-specific antibody to small beads that settle out of solution readily. Second, you lyse cells, extract the soluble proteins, add the antibodies + beads, and incubate for 2 hours. Third, you centrifuge the mixture to separate the antibodies + beads and any bound proteins from the unbound proteins. Fourth, you wash the beads three times to remove| non-specifically bound proteins. Finally, you add detergent to the beads to denature and dissociate all proteins before separating them on a gel and revealing them via immunoblotting (shown on right where \"+\" and \"-\" indicate what was or was not expressed as a soluble protein in your cells). Your partner previously hypothesized that Elm binds directly to Oak and Oak binds directly to Red, forming a \"chain.\" What do your data reveal about your partner\'s hypothesis? What is the significance of the data in the second lane? Why was this combination of proteins important to include? Your lab partner looks at your gel and says that you have definitive evidence that Elm binds directly to Oak and to Red. Do you agree or are there plausible alternative interpretations of your data? Explain.

Solution

First let us be clear with the picture

Lane 1 - Depicts that all the three components are expressed.

Lane 2 - Depicts that in the absence of Elm, neither Oak nor Red is expressed.

Lane 3 - Depicts that Elm binds with Oak and are expressed even in the absence of Red

Lane 4 - Depicts that Elm binds with Red and are expressed even in the absence of Oak

The lab partner is correct in saying that Elm binds directly to Oak and to Red. This is evident by the fact that Oak and Red are expressed even in the absence either one, provided Elm is present. This can be further witnessed in lane 2, as the absence of Elm, reveals the non expression of Oak and Red.