2D-Electrophoresis: Videos & Practice Problems

Two-dimensional electrophoresis (2D electrophoresis) combines isoelectric focusing and SDS-PAGE to separate proteins based on both isoelectric points (pI) and molecular weights. This technique effectively distinguishes proteins with identical pIs but different molecular weights, as well as those with identical molecular weights but different pIs. The first dimension uses a stable pH gradient for isoelectric focusing, while the second dimension employs SDS-PAGE to separate proteins by size, enhancing proteomic analysis and providing insights into protein characteristics.

2D-Electrophoresis

In this video, we're going to talk about 2D electrophoresis. So 2D electrophoresis or two-dimensional electrophoresis is just a combination of two electrophoresis techniques that we already talked about, and it's really just a combination of isoelectric focusing followed by SDS-PAGE in a second perpendicular direction. And so the reason it's called 2D electrophoresis is because we run isoelectric focusing, which is a type of electrophoresis, in one dimension, and then we turn our isoelectric focusing gel 90 degrees so that we can run our second electrophoresis, SDS-PAGE, in a second dimension that is perpendicular to the direction of the first dimension. And we'll be able to see how that works better down below in our example of 2D electrophoresis. Now before we get there, it's really important to note that 2D electrophoresis accomplishes two different tasks that either technique actually fails to do on their own. And so these two different tasks are essentially the advantages of using 2D electrophoresis. And so the first task that 2D electrophoresis accomplishes is that it separates proteins with identical pI's or isoelectric points, but different molecular weights. And so we know that isoelectric focusing separates proteins based on their isoelectric points. But two different proteins that have different molecular weights, one with a large molecular weight and one with a small molecular weight, if they have identical pI's or isoelectric points, they're actually going to appear as a single band if we just do isoelectric focusing on its own. But with 2D electrophoresis, we're actually able to separate those proteins that have identical pI's and different molecular weights. Now in a similar fashion, the second task that 2D electrophoresis accomplishes is that it separates proteins with identical molecular weights but different pI's. And so we know that isoelectric focusing will separate the proteins based off of their pI, so they'll already be separated, by their isoelectric points. And SDS-PAGE will separate the proteins based off of their molecular weights. And so together, when we use them in combination with one another, we're able to separate the proteins with identical weights but different pI's, which is something that SDS-PAGE is not able to do on its own. And so we'll be able to see, and understand how this works a little bit better down below in our example. And so in our example of 2D electrophoresis, what you'll notice is that it's really just a combination of two electrophoresis techniques that we already talked about. The first technique is isoelectric focusing or IEF, and the second technique is SDS-PAGE. And so to refresh our memories on isoelectric focusing, recall that there is a pH gradient with a linearly decreasing pH that is established into the isoelectric focusing gel. And this pH gradient is stable and immobile, meaning that it does not move throughout the entire process. And any particular region within the gel is going to have a very specific pH that, will not change throughout the entire process. And so we can load our proteins into the top of our gel and apply an electric field, and our proteins are going to begin to migrate through the gel until they reach the specific region of the gel where the pH in that region matches the isoelectric point of the protein or the pI of that protein. And so it results in all of these different protein bands that we see, and, the proteins are separated by their isoelectric points where the high pI's, the high isoelectric points appear at the top with the higher pH values, and the low pI's, the low isoelectric points appear towards the bottom with the low pH values in this region. And so we're separating proteins only based on isoelectric points. Now with our second step of, 2D electrophoresis, all we need to do is turn our isoelectric focusing gel 90 degrees sideways. And so you can see we can take this gel here and we can literally just turn it 90 degrees like this, and that's exactly what we see over here. Notice that what we have is our high pI, our high isoelectric point on the left, and our low isoelectric points on the right. And so, after, we turn our gel sideways, we can, apply a second gel down below, so this is an SDS-PAGE gel, and we can run the proteins from the isoelectric focusing gel down and into the SDS PAGE gel. And we know SDS-PAGE separates proteins based on their molecular weight, where proteins with a high molecular weight travel slower through the gel and appear towards the top, and proteins with low molecular weights travel faster through the gel and appear at the bottom. And so all of these different protein spots that you see represent individual separate proteins. And so all of the proteins that appear in a vertical plane like this, they all had the same isoelectric point, and that's because all of these proteins traveled together, in the isoelectric focusing gel. But when we ran 2D electrophoresis and SDS-PAGE in a second dimension, we were able to separate all of these proteins based on their molecular weights even though they had the exact same pI. And that is the advantage that we said before, separate proteins with identical pI's but different molecular weights. And so proteins that appear in the same horizontal plane, they had different pI's, but they, have the same exact molecular weight, which is why, they appear on the, y-axis over here at the same position. Actual image of a real isoelectric focusing gel. So, what you'll see is that over here on the far right, what we have is an actual image of a real isoelectric focusing gel. So you can see that all of these different spots here represent individual different proteins. And a real 2D electrophoresis gel can get a little bit messy here. So you can see we've got, some, splotches and stuff like that. But for some proteins, it's really effective in separating those proteins. And so this can be a really useful technique to separate proteins based on their pI or their isoelectric point as well as their molecular weights. And so the last thing I wanna leave you guys off with is that when we take our isoelectric focusing gel and we turn it 90 degrees sideways, we could take our isoelectric focusing gel and just turn it like this, one way, where we have the high isoelectric point on the left and the low isoelectric point on the right. But we could easily just as well take our gel and turn it the other way where we would have the high isoelectric points on the right and the low isoelectric points on the left. And so what that means is that it's really important for you guys to pay attention to how the axes are labeled on a 2D electrophoresis gel. And so keep that in mind as we move forward through, the rest of our practice problems. And so, I'll see you guys in our practice videos.

Use the results of the two-dimensional electrophoresis gel below to answer the following questions. 

A) Which protein or proteins have the highest pI value?
a. Protein a.                                    b. Proteins d & e.
b. Proteins b & c.


B) Which protein or proteins have the highest molecular weight?
a. Protein a.                                    c. Protein c.
b. Protein b.                                    d. Proteins d & e.


C) Which protein or proteins have identical molecular weights?
a. Proteins a & d.                           c. Proteins d & e.
b. Proteins b & c.                           d. None. Each has a unique weight.

Alright. So here's a 3-part practice problem broken up into parts a, b, and c. The text above states, "Use the results of the 2-dimensional electrophoresis gel below shown here to answer the following questions." In part a, it asks which protein or proteins have the highest pI value or the highest isoelectric point. We know that isoelectric focusing separates proteins based on their isoelectric point. For part a, we're only going to focus on this horizontal axis that runs from left to right. We are only focused on protein spots as they appear from left to right on the gel, and not so much from top to bottom. Notice that the gradient goes from low gradients on the left to high pH gradients on the right. Proteins with higher isoelectric points stop in the region of the gel that has higher pHs. Notice that the two proteins that have the highest pHs are going to be the ones that show up furthest to the right, which are protein spots d and e. Thus, for part a, we can indicate that option c here is the correct answer. Proteins d and e have the highest pI value. Moving on to part b, it says, which protein or proteins have the highest molecular weight? Since it's referring to molecular weight, we're going to focus on the SDS-PAGE axis, as SDS-PAGE separates proteins based on molecular weight. Larger proteins with higher molecular weights move slower through the gel and so they appear closer to the top, and proteins with smaller molecular weights move faster through the gel and appear closer to the bottom. Therefore, this protein here is the one that's closest to the top, so it moves the slowest because it has the highest molecular weight. That's protein spot b. We can indicate that option b here is the correct answer for part b. Moving on to our last part, part c, it asks which protein or proteins have identical molecular weights. Again, because it's referring to molecular weights, we're looking at the SDS-PAGE axis. Notice that there are only two proteins that lie on the same portion of the SDS-PAGE axis, and none of the other proteins have a partner that has the same molecular weight. Essentially, this means that protein spot a and protein spot d have the same molecular weight. So, that would be answer option a for part c. So we can indicate that A is correct for Part C. This concludes this practice problem, and I'll see you guys in our next video.

Which of the following is true in 2D-electrophoresis?

• A. Spots on the gel corresponds to protein subunits.
• B. The 1st step involves separating proteins by MW.
• C. SDS is necessary to separate proteins by pI.
• D. Proteins with identical pI but different MW separate.

Answer: (D)

An average protein will not be denatured by:

• A. A detergent such as sodium dodecyl sulfate (SDS).
• B. Heating to 100˚C.
• C. Iodoacetic acid.
• D. A sudden change from pH 7 to pH 13.
• E. Urea + β-mercaptoethanol.

Answer: (C)

Sketch the result of 2D gel electrophoresis on the following four proteins (see chart) & label them clearly.

So this practice problem says to sketch the result of 2D gel electrophoresis on the following 4 proteins seen in the chart below and label them clearly. Notice that this chart here has proteins A, B, C, and D, and we're given the molecular weight of each protein, the pI (isoelectric point) of each protein, as well as the specific charge of each protein at pH 6, pH 7, and pH 8. With 2D gel electrophoresis, it uses two electrophoresis techniques. First, it uses isoelectric focusing in one dimension where it separates proteins based on their isoelectric points. We have the low pHs on the far left and the high pHs on the far right, so isoelectric focusing is on the horizontal axis. After running isoelectric focusing, we turn our gel 90 degrees, and we run SDS-PAGE in the perpendicular direction. SDS-PAGE separates proteins based on their molecular weights, where proteins with larger molecular weights travel slower through the gel and remain at the top, and proteins with lower molecular weights travel faster through the gel and remain at the bottom. We're going to need this column over here with the isoelectric points of the proteins for isoelectric focusing and the molecular weight column of each protein for SDS-PAGE. The columns with the specific charges of each protein at different pHs are not needed for this practice problem. Let's start to plot our proteins. Our first protein is protein A. Protein A has a pI of 8.1. It will migrate to the region of the gel that has a pH matching its pI, just beyond the pH of 8, and on the SDS-PAGE, protein A, with a molecular weight of 62,457, will be found just above the marker of 60,000. Moving on to protein B, it has a pI of 5.69, so it will be found just below pH 6 on the isoelectric focusing, and with a molecular weight of 115,471, it will be just below the 120,000 mark on the SDS-PAGE. For protein C, which has a pI of 7.7, it will be positioned just below pH 8 and above pH 7, and with a molecular weight of 17,183, it will be just below 20,000 on the SDS-PAGE. Finally, protein D has a pI of 6.29 and will be located above pH 6. Its molecular weight of 69,366 places it close to the 70,000 mark on the SDS-PAGE. We have successfully separated and sketched the locations of the 4 proteins in our 2D gel electrophoresis simulation. 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 2D electrophoresis and how does it work?

2D electrophoresis, or two-dimensional electrophoresis, is a technique that combines isoelectric focusing (IEF) and SDS-PAGE to separate proteins based on their isoelectric points (pI) and molecular weights. In the first dimension, IEF separates proteins by their pI using a stable pH gradient. Proteins migrate until they reach a region where the pH matches their pI. The gel is then rotated 90 degrees, and SDS-PAGE is performed in the second dimension to separate proteins by size. This method allows for the separation of proteins with identical pIs but different molecular weights, and vice versa, providing a comprehensive analysis of protein mixtures.

What are the advantages of using 2D electrophoresis over other techniques?

2D electrophoresis offers several advantages over other techniques. Firstly, it can separate proteins with identical isoelectric points (pIs) but different molecular weights, which is not possible with isoelectric focusing alone. Secondly, it can distinguish proteins with identical molecular weights but different pIs, a task that SDS-PAGE alone cannot achieve. This dual separation capability enhances the resolution and accuracy of protein analysis, making it a powerful tool for proteomic studies. Additionally, 2D electrophoresis provides detailed insights into protein characteristics, aiding in the identification and characterization of complex protein mixtures.

How does isoelectric focusing work in 2D electrophoresis?

Isoelectric focusing (IEF) is the first dimension in 2D electrophoresis. It involves a gel with a stable pH gradient, where the pH decreases linearly from one end to the other. Proteins are loaded onto the gel and an electric field is applied. Each protein migrates to the region where the pH matches its isoelectric point (pI), the pH at which the protein has no net charge. At this point, the protein stops moving, resulting in separation based on pI. This step effectively resolves proteins with different pIs, setting the stage for further separation by molecular weight in the second dimension.

What is the role of SDS-PAGE in 2D electrophoresis?

SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is the second dimension in 2D electrophoresis. After isoelectric focusing, the gel is rotated 90 degrees, and SDS-PAGE is performed. SDS, a detergent, denatures proteins and imparts a uniform negative charge proportional to their length. When an electric field is applied, proteins migrate through the polyacrylamide gel based on their molecular weight. Smaller proteins travel faster and appear towards the bottom, while larger proteins move slower and remain near the top. This step separates proteins by size, complementing the pI-based separation from the first dimension.

What are the common applications of 2D electrophoresis in research?

2D electrophoresis is widely used in proteomics and molecular biology research. It is commonly employed for protein identification, characterization, and quantification in complex mixtures. Researchers use it to study protein expression patterns, post-translational modifications, and protein-protein interactions. It is also valuable in comparative proteomics, where differences in protein profiles between samples, such as healthy vs. diseased tissues, are analyzed. Additionally, 2D electrophoresis aids in the discovery of biomarkers for diseases and the development of new therapeutic targets, making it a crucial tool in biomedical research.