Western Blots

I’m learning how to western blots this week. Western blots are what we use to detect protein levels in cells and is a very common procedure in our lab. It’s not that hard but it takes 2-3 days to do one blot and there are a lot of steps to do. First, we have to collect the cells and lyse them. Most proteins are in the cytoplasm of the cell so we just have to break the cell membrane and collect what’s inside. If we do this with hundreds of thousands of cells we can collect a solution containing a detectable level of protein that we can then analyze. We then take the protein from the cells and load them in a protein gel. The gel is made from an acrylamide mix and can contain several different things. The one we use most is called a SDS PAGE gel. SDS is a type of detergent that changes the protein structure so that the three dimensional structure of each protein has no effect on how it runs in the gel. SDS also puts a negative charge on the proteins. This is key because it allows us apply a current through the gel that allows the proteins to travel down the gel toward the positive cathode. This separates the proteins by size; the larger proteins travel less distance while smaller proteins travel farther. After the gel is run, you have to transfer the proteins from the gel to a special membrane. I’m not sure what’s so special about the membrane (it just looks like a thin piece of paper) but that’s what we use. After we transfer the proteins to the membrane, we apply the primary antibody, which is protein specific so that it will only bind to one protein (or a couple of proteins if you’re unlucky). We then apply a secondary antibody which binds to the primary antibody. Attached to the secondary antibody is an enzyme called Horseradish peroxidase (it’s found in the roots of horseradishes hence the name). Horseradish peroxidase oxidizes a compound called luminol and releases light when the reaction occurs. We capture this light in a dark room with very little other light using a piece of film similar to the type that x ray images are printed on. We then have a machine that develops the films for us, similar to how people did before digital photography. We should see a small black band indicating that the protein is there if the process works.

Western blots aren’t used that often as a data collecting tool because they aren’t qualitative. You have no way of knowing how much protein you actually have; all you can do is compare your samples to other samples. You don’t get any numbers to compare. It can be very subjective. Western blots are mainly used to confirm that you have or don’t have protein in your sample. When we do a gene depletion like I mentioned in a previous post, we can check for the protein of interest by doing a western blot.  If we don’t see a very dark band compared to a control sample without the gene depletion, we can prove that our gene depletion was successful.

Our lab does westerns a lot. They aren’t too hard or too complicated but they are very important. They can be tricky sometimes depending on the protein but I haven’t run into any protein that is hard to see yet. I will at some point but hopefully not too soon.

MTT Assay Results

I got the results from my MTT assays today. I didn't see anything super interesting or relevant so I think I'm going to stop doing gene knockdowns. That's science though. Not every experiment can yield breakthrough results.

MTT Assays

Before I get into the project about mitotic bookmarking, I’m getting to a good stopping point in my main project. Right now, I’m doing a simple experiment called an MTT assay. MTT is a type of yellow salt that we can use to determine the activity of cells. In healthy cells, mitochondria inside the cell metabolize the MTT into a molecule called formazan, which is purple. We can then measure the light emitted by the sample to determine what concentration of formazan exists in the sample. If we observe more of a purple color, more healthy cells exist in our sample; the less purple it is, the fewer healthy cells there are. I’ve been using this type of experiment a lot to test different combinations of drugs. If we don’t observe much of a purple color, we know that our drugs are effective in killing cells. Right now, I’m using the same concentrations of drugs as I have before but I’m modifying the cells a little bit by deleting specific genes. To do this, we are using something called small interfering RNA (siRNA). When siRNA is released into a cell, they bind to messenger RNA, which are the intermediates in the process of making proteins from DNA. As a result, the mRNA is degraded so that no proteins are able to form, thus leading to a variety of potential cellular effects. This sounds difficult to achieve experimentally but it’s not that hard. You just mix a few solutions of reactants. It’s one of the most common things we do. 

The bad thing about doing MTT assays is that they are tedious. It involves a lot of pipetting of small volumes of solution. It can take an hour or two to fully set up a 96 well plate and add drugs to the cells. It’s kind of annoying but it’s one of the simplest experiments I’ve done. And it usually gives pretty good results, which is nice.

My Research

The research that I have been doing during the year is a lot different from the type of research that I'll be doing during the summer. My original project studies the effects of a combination of different cancer drugs on breast cancer cells. I have so far tested two drugs in combination with each other. The first is called Cisplatin, which is a very common chemotherapy drug that has been used since the late seventies. It causes DNA damage by binding to DNA and interfering with how cells divide. The other class of drug I am using in called an HDAC inhibitor. HDAC inhibitors cause proteins called histone deactylase (HDACs) to become inactivated. In a normal cell, HDAC proteins make the DNA less accessible so that genes can’t be easily transcribed into proteins. We hope that by inhibiting HDAC proteins, we can make the DNA more accessible and more susceptible to DNA damage by drugs such as cisplatin. Our initial results show that HDAC inhibitors increase sensitivity to cisplatin when the combination of both drugs is used. 

The main project that I’ll be working on deals with the structure of DNA during the time when cells divide, which is called mitosis. During normal cellular life, the DNA is relatively relaxed and DNA is being used to make proteins. During mitosis however, the DNA becomes very condensed. When it becomes condensed, DNA loses several of the biological markers that are used to mark where genes are being transcribed. However, it was previously unknown how cells remember which genes were being used to make proteins and which genes were inactive. My project studies the how cells remember/bookmark which genes were being made to make proteins and the mechanism behind it.

The project was mostly finished by a grad student (now Ph.D.) who worked in our lab but has now left. My job is to confirm her results by using a procedure called chromatin affinity purification (ChAP). This allows us to purify certain parts of DNA to determine what kind of bookmarks there may or may not be. I’ll probably go into this more in another post if I feel like it. But first, I have to write a new protocol based on our old lab protocol since the ChAP I’ll be doing is a little bit different.  

About Me

I also thought it would be a good idea to give you some background about myself before I get into the research part.

I am from a town called Centerville, Ohio which is south of Dayton. It’s only about an hour and fifteen minutes away from Ohio State so it’s not too far. Like all STEP participants, I’m about to enter my third year at the university. I’m studying molecular genetics and also have an English minor. This year, I’ll be taking mainly classes relating to my major and a few English classes. I’ll also be studying for the MCAT in the late summer and early fall with the hope of taking the MCAT in September or October. That’ll take a lot of my time so research may have to take a back seat for a month or two at the beginning of the school year. That’s about it I think, or at least it’s the important stuff.

Getting into Research

Before I get into the research that I’ll be doing this summer, I thought it would be a good idea to give you a little bit of information about myself and how I got into research. I started doing research at the beginning of my sophomore year. I knew that I had to start doing research because it is a major component of a med school or grad school application. Since most PIs (principal investigators) don’t advertise research positions, especially to undergrads, most people directly email many PIs and professors in order to find a position. This is what I did; I emailed two professors at college of medicine. I was specifically interested in doing cancer research which is why I choose to find research at the college of medicine. Most PIs will list what kind of research they’re doing on their website so you can kind of go through and pick projects that are most interesting to you. After I identified projects that I was interested in, I emailed the PI to ask if they had any openings in their lab. One of the PIs never responded to my request but I was lucky enough for the other one to have a spot open in his lab so I started working there and I’ve worked there for about a year now. I was lucky in that it only took me two emails to find a position. I know several people who had to wait a long time in order to find a research position. But Ohio State is a very good school to participate in research at because there are lots of different research opportunities in many different areas of research. I’ve never heard of anyone here not being able to find research. It just might take some time to find the right research position for you.