Western Blot Protocol

The Western blot protocol provides scientists with a means of comparing relative protein levels in different samples. Proteins are differentiated by size on an acrylamide gel and visualized on a membrane with antibodies. Learn more about how this protocol allows us to determine what proteins are present in a cell and under what conditions.
  • a cytolytic protein produced by cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells). When released, perforin polymerizes and forms pores in the plasma membrane of a target cell
  • serine proteases released by cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells) that induce programmed cell death (apoptosis) in target cells
  • a laboratory technique used to detect the presence of specific RNA molecules from a larger pool of RNA
  • a laboratory technique used to detect the presence of specific DNA sequence from a larger pool of DNA molecules
  • a technique used to assess the presence of post-translational modifications such as phosphates, lipids, or carbohydrate epitopes
  • a molecule with polar and nonpolar components that disrupts the hydrophobic/hydrophilic interactions of biological substances (e.g. breaking apart a cell membrane)
  • disrupting the molecular conformation of a protein
  • a medium for performing gel electrophoresis which contains long chains of amide groups
  • a set of protein standards used to estimate the size of experimental protein samples run during gel electrophoresis
  • a microporous substance which binds proteins
  • an aqueous solution that resists changes in pH when small amounts of acid or base are added
  • a large, “Y” shaped protein that binds directly to one protein or antigen with high specificity
  • a large, “Y” shaped protein that binds directly to the Fc region of a primary antibody
  • a chemical compound that can absorb and re-emit light

Even though you can’t see it, the cells that make up your body are doing lots of important tasks that keep you alive. Your immune cells are roaming around looking for foreign pathogens, cells in your gut are helping you digest food, and right now the cells in your brain are trying to help you pay attention to this video. For a cell to be able to accomplish important tasks like these, there is a lot that has to happen within the membrane, and scientists are really excited about uncovering what all of those internal cellular processes are. If they know how cells work on the inside, they can use this information to better treat, or potentially cure a wide range of diseases.

So, learning about the internal workings of a cell is a really important task, and at the heart of all of these processes are proteins which are the “doers” of the cell. Receptor proteins on the surface of a cell allow it to understand what is happening in the local environment. Internal signaling proteins basically play a game of telephone to transmit that signal down to the nucleus, and the cell can respond to its environment by making new proteins or even breaking down proteins that are already present into their amino acid subunits. The proteins that a cell is making at a given time can help us understand what the cell is doing or if it isn’t functioning properly and like I said before, this is really valuable information. For example, Natural Killer cells (or NK cells) are an important immune cell that roam around in our blood stream and kill cancerous or virally infected cells. They do this by releasing toxic proteins called perforin (which form pores in the membrane of a target cell) and granzymes (proteins that flood into the cell through the holes made by perforin, and cause the target cell to undergo apoptosis or programmed cell death). Some research studies have proposed that NK cells lose their ability to produce these proteins as we age, so we could better understand how NK cells age by looking at the amount of proteins like perforin and granzymes inside a cell at different points throughout a person’s life.

Okay, so proteins do important things within cells, and scientists want to understand these processes. A common way that scientists can visualize proteins in a cell is with a tool called a Western blot. And before you ask, yes there are actually Northern, Southern, and Eastern blots too. So let's talk about how a Western blot works. The general idea is that you take a cell, lyse it (or break it open) either by boiling it or adding something called a detergent so that all of the proteins come out. There are tons and tons of different proteins inside a cell at a given time, so to analyze one protein of interest we’re first going to sort all of the proteins by size, and then tag the one we’re interested in so we can visualize it.

To do that first separation step, we take our pool of proteins (some of which are actually stuck together in protein complexes) and denature them by increasing the temperature, or adding a chemical denaturant. This breaks up protein complexes, and causes all individual proteins to unfold. Another function of this denaturation step is to coat each individual protein in negative charges (I’ll explain why this is important later).

After doing this denaturing step, we load our proteins into a well at the top of an acrylamide gel. The gel is actually porous so it has lots of little holes and spaces inside for proteins to pass through. Then, we apply an electrical current across the gel which basically forces the proteins to move towards the bottom. This is why it is really important for the proteins in our sample to all have a negative charge so they are repelled by the negative charge at the top of the gel and attracted to the positive charge at the bottom of the gel.

You could imagine this process like a scene from a spy movie where a secret agent has to maneuver through a field of lasers to get to a safe on the other side of a room. Lets pretend that there are two agents trying to make it to the safe. One agent is really tall and the other is much shorter. Their heights represent the different lengths or sizes of denatured proteins we would use in a Western blot. So lets say we give our agents ten minutes to make it as far as they can across the room. We can see that the shorter person made it further towards the safe than the taller person did. This is because the taller person had to duck down and climb through the space between the lasers whereas the shorter person could squeeze through much more easily. If we didn’t put a time restriction on our agents, both of them would eventually make it to the end of the room but in the real world of doing a Western blot, that would mean that our proteins had literally fallen out of the bottom of the gel and that would be bad. The purple solution we added earlier actually served another purpose besides helping us denature our proteins. It’s also a loading dye that helps us know when to turn off the electric field that makes the proteins move. Like sneaky secret agents, we can’t actually see our proteins as they migrate through the gel, and the loading dye doesn’t stain the proteins or anything, it just flows through the gel ahead of the proteins and indicates when we should stop the current.

So when we allow our agents to move through the laser field for a set amount of time, we can see that a person’s height dictates how far they can make it towards the safe, and we can distinguish one agent from another based on their height. This is exactly what we want to do in a Western blot. Imagine that we let a huge group of secret agents all try and get across the laser field to the safe. After a certain amount of time, what we would notice is that the agents group together by height with the short people closest to the safe.

In a Western blot, we want to line up all the proteins in a cell according to their size or length because this will make it easier to identify the protein we’re looking for. We know how big our proteins are by comparing them to a protein ladder which we run alongside the proteins we got from our cell. A ladder is a solution that contains lots of proteins of known sizes so at the end of the Western blot, we’ll be able to compare our protein of interest to the ladder to determine how big it is. But, after running our proteins through the gel, we still can’t see any of them by eye, so now we need a way to detect the one we’re looking for.

So we have this gel, and we have to get the proteins from the gel onto a membrane where we can detect the protein we’re looking for. Well, we already made sure that our proteins were coated in negative charges, so we can rely on the same principal of using an electric current to move them onto the membrane. Proteins can’t just jump through space onto the membrane, so we have to make sure that the membrane and the gel are tightly sandwiched together. We use some filter paper and filter pads (which are basically sponges) to keep the gel and the membrane in close contact, and coated in a buffer solution. Then, we pack that whole sandwich into a tank filled with buffer that creates the electric field. So now that we’ve packaged all of our layers into the tank, lets just look at what happens between the gel and the membrane for a second. The proteins are attracted to the positive pole of the tank so they migrate out of the gel and get captured by the membrane. Keep in mind that the proteins are still invisible to the eye, so we need a way to visualize the protein we’re interested in.

Once we have the proteins transferred to the membrane, we’ll soak that membrane in a primary antibody that will only stick to the protein we want to look for. Antibodies are great because they are super specific. An antibody for protein A for example shouldn’t bind protein B so this gives us a way to tag our protein of interest amongst a large population of proteins on the membrane. So now we’ve attached this primary antibody to our protein, but that didn’t make it any more visible than it was before. What we do now is actually add another antibody, called a secondary antibody. This may seem a little weird to do, but the secondary antibody binds to the first one we added and helps us see where it is on the membrane. In a Western blot, there are a few different ways to find your protein or detect it on the membrane and these different methods mainly have to do with the type of secondary antibody you use. Let’s keep it simple and imagine that the secondary antibody is attached to a fluorophore, or a fluorescent molecule that glows when you shine a particular wavelength of light on it. So, now that we have our protein stuck to our antibodies, we can take the membrane and image it to see if our protein of interest is present and how much of it there is. Western blots aren’t the best tool for quantifying absolute protein abundance, but they can be useful for visually assessing relative protein abundance.

Let's go back to our example of Natural Killer cells from earlier. Say I took an NK cell from a young person and an NK cell from an older person and wanted to see how much of that perforin protein the two cells contain relative to each other. I would do my Western blot analysis where I dump proteins out of both cells (making sure to keep them separate), denature all the proteins, run the gel, attach the primary and secondary antibodies, and then I would take a picture of the membrane which excites the fluorophore on the end of the secondary antibody and allows me to visualize how much protein was present in each cell. A thicker band means more protein is present, so if I compare the band size between the young NK cell and the old NK cell I can see that the younger NK cell has more perforin protein compared to the older cell even if I don’t know exactly how much protein that is. This result supports the hypothesis that older NK cells have less perforin than young NK cells, but as science goes, there are still many more experiments to be done to really test this hypothesis in a thorough way.

So, in general, Western blots are a super useful tool for visualizing proteins in a cell. By understanding which proteins are present, and under what conditions, scientists can get a much better picture of what the cells in our body are up to and how we could use that information to design better treatments and therapies for countless diseases.

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