Tagged Ubiquitin Check

I finally got the results to check for the tagged ubiquitin and, of course, things aren’t that clear. Both my new cells and my old cells showed some tagged ubiquitin with the new cells showing slightly more. This is a slight problem because I was hoping that my old cells would not have shown any tagged ubiquitin, which would have definitely give me the answer as to why my experiment hasn’t been working. The fact that tagged ubiquitin was expressed in the old cells worries me a little because it could mean that there’s still something wrong with my ChAP procedure.

The problem with this check though is that I have no control. The main problem is that I don’t know how much of the tagged ubiquitin I should see. I could be getting a lot of tagged ubiquitin or not enough. The only thing that I know is that it’s there in some amount. It’s frustrating because the tagged ubiquitin is the most crucial part of the experiment but it’s hard to tell if it’s right or not.

My plan for the next week or so is to do another mock ChAP. I’m hoping that the change of cells makes the ChAP work this time. If not, I’m not sure what I’ll do.

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.

Mock ChAP

The results of my most recent ChAP attempt (I’m calling it a mock ChAP because it’s just an attempt to see if I can get the experiment to work right) weren’t so great. I got more of the same results. My % input was again too low. Since I checked the sonication and the experiment still didn’t work, the sonication isn’t a problem. I’m not sure what the problem is now. There are a couple things that could be going wrong with a couple of solutions I’m using. I suppose that it’s possible that some of the solutions I’m using are bad so I’m going to remake some of them. Other than that, I’ll have to be extra careful that I’m doing everything right and following the procedure exactly as I’m supposed to. I’m not sure what else could be going wrong at this point.

Sonication Check

Here are the results from my sonication experiment. This picture shown is the result of running the DNA samples on an agrose gel. Agrose is a type of sugar that polymerizes into a gel. In order to run an agrose gel, you have to load a small amount of DNA into the gel and apply a voltage through the gel. DNA is negatively charged so when the current in applied, DNA will move through the gel toward the positive terminal at the other side of the gel. Inside the gel, DNA pieces of different sizes will move at different rates, similar to how proteins move through a gel in western blots. Larger pieces of DNA move slower than smaller pieces of DNA which allows us to determine what size DNA fragments we have in our sample. 

Let me explain the picture a little bit. The far left and right columns are called DNA ladders. They contain DNA pieces of known size and serve as a marker for how far a specific piece of DNA should run. The bands at the bottom are smaller in size than the bands at the top of the gel. I should also mention that the darker the band, the more DNA there is in that particular location. Going from left to right in the other columns are the DNA samples that I took after I sonicated. Again from left to right, the samples are as follows: 3 cycles, 6 cycles, 9 cycles…up to 24 cycles of sonication. (A cycle of sonication is 30 seconds in the sonicator and one minute on ice). As you can see on the gel, in the samples where I sonicated less, the DNA is larger where as in the samples where I sonicated more, the DNA is in smaller pieces.  The results also show that I should be doing about 15 cycles of sonication to achieve small pieces of DNA.

When I did my first ChAP, I only sonicated for three cycles, which probably wasn’t ideal. However, I’m not sure if the DNA is large enough to cause any significant problems so I’m not sure that the sonication is that big of an issue in my ChAP procedure. This is frustrating because I don’t think that I have a good answer to what went wrong. Since I have the DNA already prepared, I think I’m going to continue with the ChAP protocol to see if the sonication helped to achieve a higher purification. Hopefully it works better this time, but if it doesn’t it means that sonication isn’t the problem that’s causing me to get bad results.