Successful Co-IP's

Quite awhile ago, I think I posted something about Co-Immunoprecipitation experiements. I dropped them for about a year or so as other, more pressing, experiments appeared on the horizon. Several months ago, I finally got back to them. I decided to go at them a little differently though. I am convinced that p75 signaling is very context driven. We know that it can cause cell death as well as cell proliferation. At first we were just interested in what the intracellular portion of the receptor was doing but I felt it was important to use a full length receptor and not just the intracellular domain (ICD). I was able to procure a plasmid that expressed full length p75 and have been using that to do double transfections of HEK 293 cells. What is of most interest to us is to see if p75 is interacting with Merlin. Merlin is a tumor suppressor gene that is mutated in the tumors we study. The way I do this experiment is to grow the 293 cells, introduce both Merlin and p75 to the cells, and let them grow for about 24 hours. The 293 cells transcribe and translate the plasmids and presumably process them appropriately. Then, I lyse the cells and isolate the protein. The last step is the actually Co-IP. Each protein has been engineered to be produced with a small peptide tag that readily binds to antibodies. I expose the cell lysate to very small agarose beads to which one of the antibodies is bound. The antibodies bind and immobilize the protein they recognize and theoretically, any protein that is associated with the bound protein should also be secondarily immobilized. After gentle washing to eliminate any random binding, the antibody-antigen association is broken and the eluted off the beads. Finally, I need to visualize the association. This is done with a Western Blot. The eluted proteins are run on a polyacrylamide gel, transferred to nitrocellulose and then probed with an antibody that recognizes a protein that was presumably associated with the original bead-bound protein.


 There is a lot of optimization that has to occur with these experiments. You need to ensure that you have the proper ratio of cell lysate to agarose beads. Lysate-bead incubation time and temperature have to be optimized, The types and amounts of detergents you are using in the lysis buffer and the wash buffer also need to tested. pH is important. Sometimes increasing the pH will increase antibody-protein binding but you also have to keep in mind that temperature can have a dramatic effect on pH depending on the type of buffer you are using. These are a few of the variables that need to be addressed. After countless experiments to control for all of these variables and others, I finally got some results that I trust. The good thing is that I have been able to replicate my results several times with different cells.

Western on the right was the result of a 4h lysate-bead incubation and the one on the left was done for 2h.  Each blot was loaded identically.  Lane 1 (from the right, next to the red dots) is the cell lysate, lane 2 is the unbound protein, lane 3 is a sample of the one of the washes, and lane 4 is the eluted p75 protein.

Now the challenge is to do the experiment in reverse. I just tried that today with my best guess for the conditions but it didn't work. It looks like I will need to take a few steps back and reoptimize for this interaction. If I can get it to work in reverse, I will feel pretty good about saying that Merlin and p75 associate. The question then arises as to whether the interaction is direct p75-Merlin binding or if the association is mediated by other, small adapter proteins such as paxillin.  I will begin to attack this question by performing the Co-IP as above and then labeling the eluted proteins with biotin.  I will then run the eluate on a gel and instead of probing with a specific antibody, I will probe with Streptavidin which binds tightly to biotin.  Streptavidin can be visualized as above.  These data will show me if there are other protein that are binding with p75 and Merlin.  Finally, the next big step will be to determine what those proteins are by mass spectrometry.  These experiements have the potential to shed some real light on how p75 helps to regulate cell growth.