ChAP Procedure

I’m going to try to explain how the chromatin affinity purification (ChAP) procedure works in as simple, nonscientific language as I can. Before my time in lab, someone modified an existing cell line called HeLa cells so that those cells express a modified form of the protein ubiquitin. Ubiquitin is a small common protein that is used in a wide variety of cellular processes. The protein is modified so that it contains a “tag” of five units of the amino acid histidine. The tag allows us to purify the tagged ubiquitin and separate it from us things in the cell. I’ll get into this more in a second but first the cells have to be prepared for purification. 

The first thing I do in this experiment is to do a gene depletion. The gene we are interested in depleting encodes a protein that we think is crucial in mitotic bookmarking. Without it, genes aren’t bookmarked as effectively in mitosis as they are when the protein is expressed. I do this like I mentioned before using the special HeLa cells that someone in our lab previously made. After we do the gene depletion, we block the cells in mitosis. This is critical because we want to study the effects of cells in mitosis so we need to collect only cells that are in mitosis. To do this, we use a chemical called thymine which stops the cells from replicating their DNA. Then we remove the thymidine and add a chemical called nocadazole which allows the process of mitosis to start but not finish. In this way, we get only cells that are in mitosis. 

The next step is to collect the cells and crosslink them. Crosslinking is a technique that can permanently attach a protein that is bound to DNA. In my case, we have some protein that is bound to DNA and we think that this protein is attached to ubiquitin as well. If we cross link the cells, we can attach the protein, DNA, and ubiquitin complex together much longer than it would be inside a noncrosslinked cell. This is important because it allows us to purify the DNA, protein, and ubiquitin complex via the ubiquitin tag. In future steps, we can reverse the crosslinks and analyze what DNA pieces were attached to protein that was ubiquitinated. 

After I crosslink the cells, I have to lyze them in order to release the DNA which is stored in the nucleus of the cell. We use a couple different chemicals that poke holes in the cell membranes which cause the cells to rupture and release everything that was inside of the cell. The DNA can then be collected by spinning down the sample until a DNA pellet forms at the bottom of the tube. Next comes the annoying part. Each sample has to be sonicated using a probe sonicator that we store in a cold room that’s probably around 35-40 degrees Fahrenheit. I spend close to 30 minutes in the cold room for each sample and I think I’ll have to do four samples so I’m going to get really cold. Anyways, what sonicating does is a breaks up the DNA into much smaller pieces that are easier to work with. 

After sonicating, I add special beads called avidin beads which only bind to the tagged ubiquitin, protein, DNA complex. After leaving this overnight, I can throw away whatever didn’t bind to the beads. Next, I reverse the crosslinks which leaves me with two things: one being the beads which are still attached to the tagged ubiquitin and second, the pieces of DNA that were once part of the complex. I can then collect the DNA and analyze them via a technique called quantitative polymerase chain reaction (qPCR). This process allows people to determine exactly how much DNA is in a sample. In my case, I hope to see a significant difference in the amount of DNA purified between my control sample and my gene depleted sample. 

This whole process takes roughly ten days so it takes a long time to get only a few data points. It’s long work but we should be able to get good results from the experiment and hopefully publish a paper containing the results sometime soon.