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.
2D-Electrophoresis - Online Tutor, Practice Problems & Exam Prep
2D-Electrophoresis
Video transcript
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.
Problem Transcript
Which of the following is true in 2D-electrophoresis?
An average protein will not be denatured by:
Sketch the result of 2D gel electrophoresis on the following four proteins (see chart) & label them clearly.