Affinity Chromatography - Online Tutor, Practice Problems & Exam Prep

Affinity chromatography is a sophisticated method of column chromatography that purifies proteins based on their specific binding capabilities to ligands attached to a stationary phase. The target protein binds to the ligand while other proteins are washed away with the mobile phase. To elute the bound protein, a soluble ligand with a stronger affinity can be added, enhancing specificity, though it can be costly. Alternatively, salt can be used for a more economical elution. This technique is highly effective for protein purification, making it a valuable tool in proteomics.

Affinity Chromatography

In this video, we're going to talk about affinity chromatography. So, affinity chromatography is another type of column chromatography that purifies a protein based on its affinity. And by affinity, what we really mean are the specific binding capabilities of the target protein of interest that we're trying to purify. It turns out that affinity chromatography is one of the most effective types of chromatography that we've talked about thus far, which means that it does the best job at purifying our target protein of interest, and that's a great thing. However, a negative aspect is that because affinity chromatography is one of the more sophisticated types of chromatography, it also makes it one of the more expensive types of chromatography. The stationary phase material that's packed inside an affinity chromatography column is actually covalently linked to what's known as a ligand. A ligand is just a small substance that specifically binds to a biomolecule, such as a protein, to form a complex with it. The way affinity chromatography works is that our target protein of interest that we're trying to purify is going to bind to the ligand that's stuck to the stationary phase. We know that the stationary phase does not move, which means that our protein of interest is going to remain inside of the column and all of the other proteins are not going to bind to the ligand, and they are going to be washed out of the column with the mobile phase.

Let's take a look at our example down below of affinity chromatography to clear this up. What you'll see is that we have a bunch of different columns here. Affinity chromatography is a type of column chromatography, and packed inside of the column is our stationary phase, which we know does not move throughout our entire process. The stationary phase is this blue material that's packed inside the column. When we zoom in on the stationary phase, we notice that it is covalently linked to these ligands, which are these y-shaped structures in this image. The ligands are just small molecules that will bind to a specific biomolecule of interest, which is our protein our target protein of interest represented by this pink protein here. Notice that our pink protein fits and binds perfectly to our ligand, which is attached to the stationary phase and does not move. In our beaker below, we have this big protein mixture, containing positively charged proteins in red, negatively charged proteins in blue, neutrally charged proteins in gray, and then our specific target protein of interest that we're trying to purify, represented by the triangular protein in pink.

If we take our protein mixture and pour it into our affinity chromatography column, our protein mixture is going to be at the top of the column. Inside this container at the top of our column is our mobile phase, and we know that we're going to be adding our mobile phase throughout the entire process. As we start to add the mobile phase, our proteins begin to separate. Notice that our target protein of interest, the pink proteins, have not moved very much because they bind to the specific ligands which are stuck to the stationary phase, so they don't travel through the column. Whereas, all of the other proteins move fairly quickly. As we continue to add more mobile phase, eventually, we'll get all of the other proteins to elute from the column and be removed from the column, whereas our target protein of interest is still bound to those ligands in our stationary phase, so our target protein of interest is essentially purified inside the column.

Now, how do we get these bound target proteins out of the column? How do we elute these target proteins from the column? What we can do is add a bound target or salt to the mobile phase. Up here in our image, we are adding a soluble ligand to the mobile phase. The soluble ligand is similar to the ligands over here, except it's a green-colored ligand, and this ligand has a stronger attraction to the target protein. When we put it inside the mobile phase and we run the mobile phase through the column, it's going to grab on to all of the target protein and eventually what we're going to get is all of our target protein eluted at the bottom and our target protein is attached to the soluble ligand that we put into the mobile phase. If we wanted to remove our target protein from the ligand, then we could go ahead and add a little bit of salt to decrease the strength of the interactions between the protein and the ligand.

There's actually an advantage to using soluble ligands versus using salt to elute our protein. By using a soluble ligand that has a specific binding affinity to our protein, that actually adds another level of specificity to our purification process. It helps to ensure that we're really purifying our protein because we're adding another level of specification by adding a ligand that's specific to our target protein of interest. That means that we have more confidence that our protein is going to be purified. But a downside to using soluble ligands is that they can be quite expensive. So, if we're looking for a more economical way to elute our proteins, then we could just use salt to elute our protein. This concludes our lesson on affinity chromatography, and we'll be able to get a lot more practice utilizing all these concepts in our practice video. So, I'll see you guys there.

In your own words, describe the principles involved in protein purification by affinity chromatography.

Alright. So this practice problem says, in your own words, describe the principles involved in protein purification by affinity chromatography. And this is actually a great practice problem because it's challenging you to take your biochemistry game to the next level. Because anytime you can describe a biochemical process in your own words, it means that you truly understand how that process works. So, if you haven't already, take some time. How would you describe affinity chromatography? Alright. Well, the way that I would describe it is that it is literally just a technique. So, affinity chromatography is a technique, and essentially what it does is it separates proteins. And so, it's a technique that separates proteins, but even more specifically than that, it separates proteins based on their binding affinities. And by binding affinities, really what we mean are just attractions. So, the binding affinities are essentially just attractions. And so, different proteins have different levels of attractions to different molecules, and those molecules that the proteins are attracted to are called ligands. And so with affinity chromatography, essentially what we do is, we use a ligand in the stationary phase. And the stationary ligand that we use is specific to our target protein of interest. And so what that means is that our target protein of interest is going to bind to that ligand, and the target protein is ultimately going to stay inside of the column while other proteins are washed out of the column, and we get rid of them. And so, essentially, the target protein can later be collected and is now pure. So, that’s essentially how affinity chromatography works. It's really important to take the time and think about how would you describe these processes in your own words. If you're able to do that, it's a good sign that you understand the process. So that concludes this practice, and I'll see you guys in our next video.

The target protein to be purified is likely eluted from the affinity chromatography column by _______________. Explain potential advantages & disadvantages of the elution strategies.

• A. Altering the pH of the mobile phase.
• B. Addition of a chaotropic agent such as urea.
• C. Addition of salt and/or free ligand.
• D. Raising the temperature in the column.

A biochemist is attempting to separate a DNA-binding protein (protein X) from other proteins in solution (proteins A, B & C). Consider the chart & answer the questions below about what type of technique is best for separation.

A. What type of chromatography is best for separating protein X from protein A? ___________________________

B. What type of chromatography is best for separating protein X from protein B? ___________________________

C. What type of chromatography is best for separating protein X from protein C? __________________________

So this practice problem says, a biochemist is attempting to separate a DNA-binding protein, protein X, from all of the other proteins in solution, which are proteins A, B, and C. Consider the chart and answer the questions below about what type of technique is best for separation. Notice that the chart has information for proteins A, B, C, and protein X, and we're given the pI or the isoelectric point as well as the molar mass for each of the proteins, and we're also told whether or not the protein binds to DNA. If the protein binds to DNA, it means that the protein has an affinity for DNA, and that means that DNA could potentially be used as a ligand in affinity chromatography. Keep that in mind as we move along. Now, part A asks, what type of chromatography is best for separating protein X from protein A? We're only going to be focusing on protein X and protein A. Notice, looking at the isoelectric point, that both protein X and protein A have about a similar isoelectric point. The isoelectric point of protein X is 7.8, and the isoelectric point of protein A is 7.4. At about the same pH, around 7.6, both protein A and protein X are going to have about a neutral net charge. If they have about the same net charge, using the charge as a means for separating them is not a good idea, and ion exchange chromatography is not a good idea here. Now, looking at the binding to DNA, notice that both protein A and protein X bind to DNA. Using DNA as a ligand in affinity chromatography is not a good idea to separate these two proteins. Looking at the molar mass, notice that protein A has a molar mass of 82,000, which is almost four times larger than the mass of protein X. What this means is that these two proteins differ greatly in their mass and size. Using size as a means for separating these two proteins is going to be a good idea since protein A is much larger than protein X. Size exclusion chromatography is probably going to be the best chromatography technique to separate these two. So down below, we can put size exclusion. Now looking at part B, it asks, what type of chromatography is best for separating protein X from protein B? Again, we're only going to be focusing on proteins B and X. Notice this time, the molar mass of protein B is about the same as the molar mass of protein X. That means that they're about the same size, and using size exclusion chromatography this time is probably not going to be the best idea to separate them. Again, looking at their DNA binding, both proteins B and X bind to DNA. Using DNA as a ligand in affinity chromatography, again, is not a good idea to separate these two proteins. Now looking at the isoelectric point this time, notice that the isoelectric point of protein X is about double that of the isoelectric point of protein B. What this means is that at any pH here, proteins B and X are going to differ greatly in their charges. Using charges as a means for separating these proteins is going to be a good idea. Ion exchange chromatography is the type we want to use. So down below, we can put ion exchange chromatography. Now looking at part C, it asks what type of chromatography is best for separating protein X from protein C. We're going to be focused on comparing proteins X and C. This time, notice that they both have about the same isoelectric point, which means that at a pH around the isoelectric point, they're both going to have about a neutral net charge. If they have the same net charge, then using the charge as a means for separating them is not a great idea. So, ion exchange chromatography is not going to be a good idea. Again, looking at their mass, notice that they have about the same mass. That means they're going to have about the same size, and using size exclusion chromatography is not a good idea. But when we look at their DNA-binding capabilities, notice that protein X does bind to DNA, but protein C does not bind to DNA, which means that using DNA as a ligand in affinity chromatography is probably going to be a good way to separate protein X from protein C. So, down below, we can put affinity for the type of chromatography that would be best for separating them. This concludes our practice problem, and I'll see you guys in our next video.

Here’s what students ask on this topic:

What is affinity chromatography and how does it work?

Affinity chromatography is a type of column chromatography used to purify proteins based on their specific binding capabilities to ligands attached to a stationary phase. The stationary phase is covalently linked to ligands, which are small molecules that bind specifically to the target protein. When a protein mixture is passed through the column, the target protein binds to the ligands, while other proteins are washed away with the mobile phase. To elute the bound protein, a soluble ligand with a stronger affinity can be added, or salt can be used to disrupt the protein-ligand interaction, allowing the purified protein to be collected.

What are the advantages and disadvantages of using affinity chromatography?

Affinity chromatography is highly effective for protein purification due to its specificity, as it targets proteins based on their unique binding capabilities to ligands. This results in a high degree of purity for the target protein. However, the technique is sophisticated and can be expensive, particularly due to the cost of ligands. Additionally, the method requires careful optimization to ensure that the target protein binds effectively to the ligand and is subsequently eluted without losing its activity.

How do you elute a target protein from an affinity chromatography column?

To elute a target protein from an affinity chromatography column, you can add a soluble ligand with a stronger affinity for the target protein to the mobile phase. This ligand competes with the stationary phase ligand, binding to the target protein and allowing it to be washed out of the column. Alternatively, you can use salt to disrupt the interaction between the protein and the ligand, causing the protein to elute. Using a soluble ligand enhances specificity but can be costly, while using salt is more economical.

What is the role of ligands in affinity chromatography?

In affinity chromatography, ligands play a crucial role by specifically binding to the target protein of interest. These ligands are covalently attached to the stationary phase within the column. When a protein mixture is introduced, the target protein binds to the ligands, while other proteins are washed away with the mobile phase. This selective binding allows for the purification of the target protein. Ligands can be small molecules, peptides, or antibodies, and their specificity is key to the effectiveness of the purification process.

Why is affinity chromatography considered one of the most effective types of chromatography?

Affinity chromatography is considered one of the most effective types of chromatography because it leverages the specific binding interactions between a target protein and a ligand. This high specificity allows for the selective purification of the target protein from a complex mixture, resulting in a high degree of purity. The method's ability to isolate proteins based on their unique binding properties makes it highly efficient and reliable for protein purification, which is particularly valuable in proteomics and other biochemical applications.