Hi there, I'm Jamie Dyer. This is a turtle. Can you tell what sex it is? Yeah, me either. But in a recently published paper, scientists identified a new way to determine the sex of baby turtles using a western plot, which it turns out can be really helpful for conservation efforts. Now, you might think that determining the sex of a baby turtle should be easy, right? Just look up at skirt. But looking at their genitals only works for sexually mature adult turtles. So, I mean, couldn't you just look at their sex chromosomes? You probably know that for humans, females have two X chromosomes and males have an X and a Y chromosome. So why not just analyze the sex chromosomes of the baby turtles? Well, that won't work because most species of turtle don't have sex chromosomes. What? It turns out what determines whether a turtle becomes male or female is the temperature of the nest when the eggs are incubating. For most species, warmer nest temperatures result in females and cooler temperature result in males. This makes climate change especially problematic for endangered species, because as temperatures rise, an imbalance in the male to female ratio can make it harder for turtles to reproduce, which could lead to extinction. So, to try to save endangered turtle species, scientists need a way to non-invasively monitor the sex ratio of baby turtles on a large scale. And the scientists who published this school paper came up with a way to do that using a western plot. So, essentially, a western plot is just gel electrophoresis for proteins instead of DNA. Now, if you don't know what gel electrophoresis is, and the rest of this video probably won't make sense. So, go check out this video for more information. Okay, so a western plot is a lot like gel electrophoresis. Scientists use an electric current to separate proteins on a gel by size, but to see the proteins in the gel, they do something different than what they do for a DNA gel. Instead of using a dye that binds to DNA, they use antibodies that bind specifically to a protein of interest, to tell whether and how much of that protein is present in a sample. So, the scientists studying baby turtles used a western plot to determine if a certain protein was present in the blood of the baby turtles. Even though their genitals aren't well developed yet, male and female turtles have different hormones circulating in their blood. Now, not all hormones are proteins, but one sex-specific hormone in turtles called anti-malarion hormone is a protein, which means the scientists could detect it using a western plot. By looking to see whether AMH was present in their blood, the scientists could tell which baby turtles were males and which were females. Here is the western plot from their paper. Interpreting a western plot is essentially the same as interpreting a DNA gel from gel electrophoresis. Bands higher up in the gel represent bigger proteins and fatter bands represent samples that have more protein. But even though you interpret a western plot the same way you interpret gel electrophoresis, how the bands were visualized is very different. And to understand that difference, you need to understand how a western plot works. There are three main steps to doing a western plot. Electrophoresis, transfer, and blotting. The first step, electrophoresis, is essentially the same as general electrophoresis, except that it's called sodium-dodecl-doducyl-doducyl-doducyl. Sodium-doducyl sulfate polyacrylamide gel electrophoresis. It's usually just shortened to SDS page. It's just gel electrophoresis, except instead of making the gel out of agarose, which they do for DNA, they make the gel out of polyacrylamide. And the SDS part of SDS page comes from the name of the detergent that scientists add to the protein sample before they load it on the gel. See, only negatively charged molecules will move through the gel toward the positive pole, but not all proteins are negatively charged. So scientists add SDS to the protein sample, because SDS is negatively charged, and it sticks to all types of proteins and unfolds them, making sure that each protein is negatively charged long chain of amino acids that can snake through the pores of the gel. Once the gel is done running, you can't just see the proteins, they're tiny molecules. Now, if you were running a DNA gel, at this point you would just add a dye that binds to DNA and look to see where the bands are. And actually, you can do that with an SDS page gel. It's called doing a kumasi stain, but it will show you all of the proteins in the sample, not just the one you're looking for. In the baby turtle study, the scientists ran a sample of blood on an SDS page gel, and well, blood has a lot of proteins in it. So how do the scientists know which of these bands is the AMH protein? That's where the next two steps of a western block come in. Remember that the goal of a western block is to visualize specific proteins. To do that, scientists use antibodies, like literally the part of your immune system that recognizes invaders. It turns out that the mammalian immune system is really good at creating protein antibodies that bind specifically to foreign proteins. But where do scientists get an antibody that bind specifically to AMH protein? Well, they injected a little bit of purified AMH protein into a mouse, or a rabbit, or a goat. For this paper, it was a mouse. So that mouse mounted an immune response and started making antibodies against AMH. Then scientists harvested those antibodies and used them to visualize AMH protein in the western block. But the problem is that antibodies themselves are proteins, which means you can't just squirt them onto the polycrylamide gel and hope that they can get in there. And this is where the transfer step comes in. To get the proteins out of the gel, but to keep them in this nice organized order, scientists use an electric current to transfer the proteins from the gel to a thin plastic membrane, which holds the proteins in place while making them accessible to the antibodies. Finally, we're at the blotting part of the western block. This is the point where the scientists squirt the antibodies on the membrane to bind directly to AMH protein. So now we have this membrane with antibodies stuck just to the AMH protein. But you can't actually see anything yet, right? Antibodies are molecules. You can't see them by eye. So we need a way to see where the antibodies are. And for that, we use another kind of antibody. We call the first one, the one that binds to our protein of interest a primary antibody. Secondary antibodies bind to the primary antibody. Literally a biotech company injected a goat with mouse antibodies, and the goat produced anti-mouse antibodies. But before selling those anti-mouse antibodies, the company modified them, either with a fluorescent molecule or an enzyme that converts a chemical reagent into a colored product. So secondary antibodies give you a way to actually see where your protein of interest is on the membrane. And because multiple secondary antibodies bind to each primary antibody, they help amplify the signal. Then, all the scientists have to do is take a picture of the membrane with a special camera. And finally, after all those steps, they can see their western blood. Now, you may be wondering why they call it a western blood. It's actually a play on words. Back in the 70s, a scientist named Edwin Southern invented a method of detecting specific pieces of DNA by running a gel and blotting afterward. Scientists called that method a southern blood, named after Edwin. When scientists started running protein on gel and blotting for it, they called it a western blood. Actually, there's a northern blood too, but not an eastern blood. Hmm. Also, just say you know, sometimes scientists will call a western blood an immunoblot, or immunoasse, the immuno referring to the antibodies that are used during the blotting step. But they all mean the same thing. So, the scientists who are studying the sex of baby turtles, they use a western blood to determine whether AMH protein was present in the baby turtles blood. They found that AMH protein was present in 100% of one-day-old male turtles, but not in one-day-old female turtles, which they verified using laproscopy to determine the sex of the turtles when they were older. Having a non-invasive way to monitor sex ratios of baby turtles can be an important tool in conservation efforts for endangered species, all with the help of western blood. Now, that's cool.