2nd ChAP Results

My second real ChAP results were a little bit different but still not correct. I did achieve higher levels of % input, closer to what they should be. The problem is that my negative control, a gene called IL2, was also very high. A negative control is used to show that things you expect not to work don’t actually work. It’s a way to avoid getting false positives. In my case, I likely got a false positive because my IL2 was high. In the type of cells I’m using, IL2 is not expressed so you would expect to see a low level of IL2 bookmarking. Since I observed a higher IL2 amount than I should have, something went wrong with the experiment and my results aren’t reliable. This is a different kind of error than what has happened before. We think that there could be something wrong with the cells since IL2 bookmarking was so high, which shouldn’t be the case. However, my % input was higher than normal, which I’m having a hard time of explaining. 

There are a couple things that could be wrong with the cells. First, the cells could just be old. Although the cells we are using grow forever, we do find that weird things start happening when we grow them for a long time, such as the expression of a gene that is not normally supposed to be expressed. This is a possibility and we don’t have any real way of testing this. To eliminate this possibility I’m thawing new cells that I know come from a stock that work. The other possibility that I can think of is that the ubiquitin tag isn’t being expressed that well. Again, I have no idea why this would be but it would explain why I’m not getting a very high % input. We can test this however. What we can do is run a western blot and use an antibody like molecule that binds to a molecule called avidin which binds to the tagged ubiquitin (the antibody like molecule is actually a protein with horse radish peroxide that binds to avidin). We should see a streak at the top of the gel, indicating the presence of tagged ubiquitin. 

In this case, I’m actually hoping that we find that I’m not using cells with tagged ubiquitin. Although it would mean I had wasted a lot of time with a simple issue, I would finally know what was going wrong with my experiments and maybe finally finish this project.