in this video, we're gonna talk about two D electro freezes. So to the 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 ISO electric focusing, followed by S. D s page in a second perpendicular direction. And so the reason it's called two D electrophoresis is because we run ISO electric focusing, which is a type of electrophoresis in one dimension. And then we turn our ISO electric focusing gel 90 degrees so that we can run our second electrophoresis SDS page and 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 two D electrophoresis. Now, before we get there, it's really important to note that two D 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 two D electrophoresis. And so the first task that to the electrophoresis accomplishes is that it separates proteins with identical P eyes or is electric points, but different molecular weights, and so we know that is a electric. Focusing separates proteins based on. There is a electric 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 P ice or is electric points, they're actually going to appear as a single band If we just do. Is electric focusing on its own but with two D electrophoresis were actually able to separate those proteins that have identical P ice and different molecular weights. Now in a similar fashion Thio, the second task that to the electrophoresis accomplishes is that it separates proteins with identical molecular weights but different P eyes. And so we know that is electric. Focusing will separate the proteins based off of their P I, so they'll already be separated by. There is electric points, and STS Page will separate the proteins based off of their molecular weights. And so together when we use them in combination with one another, were able to separate the proteins with identical weights but different P eyes, which is something that s DS pages not able to do on its own and so we'll be able to see on understand how this works a little bit better down below in our example. And so in our example of two d electrophoresis, which will notice, is that it's really just a combination of two electrophoresis techniques that we already talked about. The first technique is is so electric focusing or I e f. And the second technique is S D s page. And so to refresh our memories on is electric focusing. Recall that there is a pH Grady int with a linearly decreasing pH that is established into the is electric focusing gel. And this pH Grady int is stable and immobile, meaning that it does not move throughout the entire process. And any particular region within the gel is gonna have a very specific pH that will not change throughout the entire process. And so we can load, are proteins into the top of our gel and apply an electric field, and are proteins are going to begin toe migrate through the gel until they reach the specific region of the gel where the pH in that region matches the iso electric point of the protein or the P I of that protein. And so it results in all of these different protein bands that we see and the proteins air separated by There is a electric points where the high p I is the high isil Electric points appear at the top with the higher pH values and the low p I is the low is electric points appeared towards the bottom with the low pH values in this region. And so we're separating proteins Onley based on ISO electric points. Now, with our second step of to the electrophoresis, all we need to do is turn our is electric 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 P i r high. I saw electric point on the left and are low is electric points on the right. And so, uh, after we turn our gel sideways, we can apply a second gel down below. So this is an S D s page gel and we can run the proteins from the ISO electric focusing gel down and into the ice. The STS page Joe and we know STS 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 jail 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 ISO electric point. And that's because all of these proteins traveled together, uh, in the ISO electric focusing gel. But when we ran to the electrophoresis and STS 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 P I. And that is the advantage that we said before separate proteins with identical P ice but different molecular weights and so proteins that appear in the same horizontal plane. They had different p ice, but they have the same exact molecular weight, which is why they appear on the Y axis over here at the same position. And so what this means is that we're able to separate proteins that have identical molecular weights, which would be these proteins here that lined up horizontally but different p ice. And so what you'll see is that over here on the far right, what we have is an actual image of a really ISO electric focusing gel, so you can see that all of these different spots here represent individual, different proteins, and a rial to D electrophoresis gel can get a little bit messy here. So you can see we've got some, uh, splotches and stuff like that. But for some proteins, it's really effective and separating those proteins. And so this could be a really useful technique to separate proteins based on their p I or there is a electric point as well as their molecular weights. And so the last thing I want to leave you guys off with is that when we take our is electric focusing gel and we turn it 90 degrees sideways, we could take our is electric focusing gel and just turn it like this one way, where we have the high ice electric point on the left and the low is electric point on the right, but we could easily Justus well, take our gel and turn it the other way where we would have the high isil electric points on the right and the low is electric 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 two D 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.
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Which of the following is true in 2D-electrophoresis?
Spots on the gel corresponds to protein subunits.
The 1st step involves separating proteins by MW.
SDS is necessary to separate proteins by pI.
Proteins with identical pI but different MW separate.
An average protein will not be denatured by:
A detergent such as sodium dodecyl sulfate (SDS).
Heating to 100˚C.
A sudden change from pH 7 to pH 13.
Urea + β-mercaptoethanol.
Sketch the result of 2D gel electrophoresis on the following four proteins (see chart) & label them clearly.