in this video, we're gonna talk about ISO Electric focusing so I so electric focusing can be abbreviated with the three letters E f. And that, of course, stands for ISO Electric focusing. And so I saw Electric Focusing is really just a type of electro for Reese's technique, which means that it uses an electric field to separate proteins and, as indicated by the word, is electric. Here, proteins are Onley separated based on their ISO electric points, and recall from our previous lesson videos that the is electric point can be abbreviated with letters P I and the P I or the Is Electric Point is really just a specific pH where the net charge of the protein is equal to zero, which means that the protein is gonna have an overall neutral net charge when the P H is equal to the YSL Electric point. So the way that I so electric focusing works is that there's a stable or a mobile pH Grady int that is established into the gel. And so let's take a look at our example down below to clear up this idea. And so again, with is electric focusing, which will notice his own Over here on the far left, what we have is a container that contains our ISO electric focusing gel. And what's important to note is that within this gel is a linearly decreasing pH Grady int, where the pH is starting high at the top of the gel. And as you move down towards the bottom of the gel, the pH decreases in a linear fashion until it gets to the Ph of three, which is at the very bottom of the jail. And it's also important to note that by the word and mobile, what we really mean is that the pH does not move throughout the entire process. And so what that means is that at this point in the gel, the pH is going to be nine throughout the entire process. At this point in the gel, the pH will be five throughout the entire process and at the bottom. The pH is going to be three throughout the entire process. And so that is exactly what we mean by a stable and in a mobile pH. Grady int that does not move, Even though the proteins will move through the gel, the pH stays the same in any particular region throughout the entire process. Now proteins are actually going to alter their charges as they migrate through different regions of the gel that have different pH is. And that's because we know that by changing the pH that will affect the ionization of the ionized able groups because the P. K s air going to stay the same, but the pH is will change, and that will change the ionization states of the Ionized Herbal Group. And that will change the charges of the protein as they migrate through different regions of the gel with different pH. And so the important part of is electric focusing is that proteins are actually going to continue to migrate until they reach the specific portion of the gel that has a pH that is equal to the P I or the is electric point. And so we all know from our previous lessons that when the pH of the solution is equal to the P I of a protein that protein is going toe have a neutral net charge of zero, and when the neutral when a protein has a neutral net charge of zero, then it will not migrate in an electric field, and that means that it's going to stop moving at that particular region inside of the gel. And so let's take a look at our example down below to see how this works. So notice that we are loading our protein sample here at the very top of our eyes electric focusing gel. And so, uh, we're going to apply an electric field, and the proteins are going to begin to move through the gel and move through the different regions of the gel that have different pH is so the proteins are going to continue to move through the regions of the gel until they get to the specific region of the gel that where the P H is equal to the P I of that protein and then the protein is going to stop, um, right at that region. And it will not move through the Joe anymore because the protein has a neutral net charge and proteins with neutral net charges do not migrate in an electric field. And so what you can see is the result is we have all of these different protein bands that are stopping exactly where their P H is equal to the piat. And so what we can do is say, for this particular protein band that's here. Whatever pH this region corresponds to and are Grady int, that would be the p I of this particular protein. And so, by just knowing that the pH. Grady in is decreasing in a linear fashion, we can determine all of the is electric points of all the proteins based on where they stop moving in our gel. And so, again, at the top of our gel, we're going to have a high P i or high is electric point because it corresponds with higher pH values. And towards the bottom of our gel, we're going toe have low p ice or low ISO electric points because these proteins here, uh, stopped at a region with a lower pH. And so the biggest take away that you guys should know about is electric. Focusing is that proteins migrate until they reach the specific region of the gel where the P H is equal to the P I and, uh is electric. Focusing separates proteins based off of their ISO electric points. And so this concludes our lesson on is electric focusing, and we'll be able to get a little bit of practice in our next couple of videos, so I'll see you guys there.
At some point during isoelectric focusing, proteins stop moving through the gel because:
The proteins do not have ionized groups at that pH.
The proteins have a net charge of zero at that pH.
The proteins have a net positive or net negative charge at that pH.
Their mass is too large to be moved at that position in the gel.
Mark the approximate final position of the following tripeptide on the isoelectric focusing gel: Glu-Met-Asp.
Hint: calculate the isoelectric point of the peptide.