Hi. In this video, I'm going to be talking about studying proteins. So scientists, of course, have a variety of different techniques that they use to study protein structure and function, and just proteins in general. So I'm going to go through a few of those techniques and just briefly describe, you know, what they're used for.
So the first is the SDS-PAGE or polyacrylamide gel electrophoresis, shortened SDS-PAGE. And this is used to detect proteins in a certain sample. And so what you do is you get a protein solution, variety of different ways to do that, and then you have a gel that's like hard or it's kind of flexible. It's kind of squishy material made out of polyacrylamide, and this is just a matrix. So if you were to zoom in real close into it, what it would look like is it would have a bunch of pores in it. And you can adjust these pores, you can make them bigger, and you can make them smaller. But essentially when proteins flow through those pores, it's going to be hard, right? They're going to get stuck, sometimes they won't fit, it's going to be really hard for them to get through. So if the pores are bigger, you know, they migrate easier, but if the pores are smaller, they migrate harder or slower. And so, essentially, what happens is you take these proteins in a protein solution, you incubate them with this thing called the SDS, it's a detergent. You'll find it in, you know, your dishwashing detergent, your laundry detergent, and that SDS attaches to the protein everywhere. And the reason it does this is for a lot of different reasons, but what the purpose of it is is because it masks the protein charge, so proteins have charges. But we don't want to sort these based on charge, we want to sort these based on size. And so, the SDS makes all the proteins negatively charged. In that way, when you run a charge through it, the proteins are all going to head towards the positive charge at the same rate. So when you run that gel, you put a protein solution, made all the proteins negative by interacting with SDS, and then you run that charge through it and they all migrate through the pores in the acrylamide mixture towards the positive charge. Then once you have that gel you transfer that, so you take that protein from the gel and you transfer it onto some type of filter, or paper, nitrocellulose paper for instance. And you do this again, they're in an electric field, but this time the electric field is going from the gel to the paper instead of through the gel. And then, eventually, you have this piece of paper with your proteins on it, and they're separated by size. So then you can take antibodies for a specific protein, for instance, and you incubate them in a process called immunoblotting. So just sort of putting an antibody solution on this paper, and the antibodies will bind to the protein if the protein is there. So then you can look at that, I mean say, well, is my protein here because did the antibody bind to it. So that's one way to look at proteins.
And then the second one, it's used less frequently, but it's called 2D gel electrophoresis. And this is awesome because you can actually detect up to 2000 proteins on the same membrane. So the negative or the downside to the SDS-PAGE is that you have you know have to use an antibody. You can use one, you can use two, but essentially you have to know the protein you're looking for. But 2D gel electrophoresis is actually detect up to 2000 at the same time. And instead of just sorting on charge or separating by charge, they're also sorted based on pH, which sort of gives this diversity of separation that allows you to detect so many proteins. So this is what an SDS-PAGE looks like. You can see that I have protein here, protein a, protein b, and I run it on some kind of gel. So the negative charge would be here and the positive charge would be here. So when I put protein right up here and run this electrical current through it, the protein is going to migrate through all the pores. Now, larger proteins are going to run slower, so they're going to travel slower and be higher because they're larger in size. And smaller proteins are going to run faster because they could easily get through those pores or more easily get to the pores than the larger protein. So they're going to run, slower or faster because they're smaller in size. So faster equals smaller, and slower, the migration is slower equals bigger. So this one here is going to migrate slower because it's bigger, and this one will migrate faster because it's smaller.
So those are some gel techniques for proteins. Then, I want to talk about other ways to add. So these are things that, you know, you're looking at certain proteins, but sometimes you don't know what these proteins are or what they're doing. So mass spectroscopy is a technique used to identify unknown proteins. It does other things too, but generally, you know, a lot of people use mass spectroscopy to do that. And so pretty much what you do is you have samples, that contain proteins or peptides, so short, amino acid segments. And so the samples contain the peptides and sort them via a mass to charge ratio. And so, they get this they end up with this graph, and I'm going to show you, it kind of looks like this. But they use this data which tells them about, you know, what the size is and what the charge is to get the sequence of the peptide. And then those peptide sequences, once you have them, can actually just be put into some software, and that software will tell you, oh, this is from this protein, or, this is from this organism, or, this is from this organism, or, you know, things like that. That's what spectroscopy is used for. Then we have, NMR, which you'll talk about more in chemistry classes, but these can be used to analyze structures of proteins. And then if you want to know if 2 proteins are interacting or interacting, one of the best ways is the yeast two-hybrid system. And so this is able to examine if 2 proteins interact inside a living organism. This is a super important part. So there's a lot of techniques to look at interactions outside of a living organism, but then you question, oh, is that real or is it just because they're sitting so close together in a tube? So this actually looks at inside a living organism. So what you happen is you have 2 proteins of interest called bait or prey, and then you fuse these two transcription factors. That way, if the bait and prey proteins are interacting in the cell, then the transcription factors will interact, and then the transcription factor interaction will actually transcribe a gene. So what this looks like is you have these two proteins. These are the ones you're saying, do these interact? In this case, they are. So you have protein 1 and protein 2 or bait and prey, and they're interacting, but they're also fused here to transcription factors. Now, these transcription factors interact because the bait and prey interact, then this is going to support the transcription of some kind of reporter gene. So that is the yeast two-hybrid system, and it says, you know, these 2 proteins are interacting. These 2 proteins are going to interact, and that's going to transcribe this gene. So those are a few techniques used to study protein. So with that, let's now move on.
