Isoelectric Focusing - Online Tutor, Practice Problems & Exam Prep

Isoelectric focusing (IEF) is an electrophoresis technique that separates proteins based on their isoelectric points (pI), the pH at which a protein has a net charge of zero. A stable pH gradient is established in the gel, allowing proteins to migrate until they reach a region where the pH equals their pI, at which point they stop moving. This results in distinct protein bands, enabling the determination of their pI values. Understanding IEF is crucial for applications in proteomics and protein analysis.

Isoelectric Focusing

In this video, we're going to talk about isoelectric focusing. Isoelectric focusing can be abbreviated as IEF, which stands for isoelectric focusing. Isoelectric focusing is a type of electrophoresis technique, which uses an electric field to separate proteins. As indicated by the word isoelectric, proteins are separated based on their isoelectric points. Recall from our previous lesson videos that the isoelectric point can be abbreviated as pI, and the pI (isoelectric point) is a specific pH where the net charge of the protein is equal to zero. This means that the protein will have an overall neutral net charge when the pH is equal to the isoelectric point.

How isoelectric focusing works: A stable or immobile pH gradient is established in the gel. In our example, notice to the far left, there is a container that contains our isoelectric focusing gel. It's important to note that within this gel, there is a linearly decreasing pH gradient, where the pH starts high at the top of the gel, and as you move down, the pH decreases linearly until it reaches the pH of 3 at the very bottom. By the term immobile, we mean that the pH does not move throughout the entire process. Thus, at different points in the gel, the pH is constant throughout the process: pH 9, pH 5, and pH 3, respectively, from top to bottom.

During the process, proteins alter their charges as they migrate through regions of the gel with different pH values. This is because the ionization states of ionizable groups change with pH, while the pKa values remain constant. Proteins will continue to migrate until reaching the specific portion of the gel where the pH equals their pI. At this point, when a protein's pH matches its pI, it will have a neutral net charge of zero and, consequently, will stop moving in the electric field.

Let's look at how this works in practice. We load our protein sample at the top of our isoelectric focusing gel and apply an electric field. The proteins begin to move through the gel, navigating through regions with varying pH levels until each protein reaches the region where the pH matches its pI. The protein then stops moving because it possesses a neutral net charge. As a result, we observe distinct protein bands, each stopping exactly where the pH equals the pI. By knowing that the pH gradient decreases linearly, we can determine the isoelectric points of all the proteins based on where they stop moving in the gel. Towards the top, we have proteins with high pIs corresponding to higher pH values, and towards the bottom, we have proteins with low pIs stopping at regions with lower pH.

The key takeaway from isoelectric focusing is that proteins migrate until they reach the region of the gel where the pH matches their pI, effectively separating them. We'll have an opportunity for practice in our next few videos. I'll see you guys there.

At some point during isoelectric focusing, proteins stop moving through the gel because:

• A. The proteins do not have ionized groups at that pH.
• B. The proteins have a net charge of zero at that pH.
• C. The proteins have a net positive or net negative charge at that pH.
• D. 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. 

So this video is really going to bring back a lot of great memories from our previous lesson videos, and that's because we're going to need to remember how to calculate the isoelectric point of a peptide. And so if you don't remember how to calculate the isoelectric point of a peptide, make sure you go back to rewatch those lesson videos really quick before you continue here. And so this practice problem wants us to mark the approximate final position of the following tripeptide on an isoelectric focusing gel, and the tripeptide is glutamic acid, methionine, and aspartic acid. And so recall from our previous lesson video that isoelectric focusing is an electrophoretic technique that separates proteins based on their isoelectric points. And so the hint tells us to calculate the isoelectric point of the peptide. And notice that we have an isoelectric focusing gel provided over here on the right. And so, we need to mark the position that this peptide is going to end up in after we run isoelectric focusing. Okay. So first, let's calculate the isoelectric point of this peptide. And so recall from our previous lesson videos that the first step in calculating the isoelectric point of a peptide is just to know how the ionizable groups of the amino acids ionize. And so, what that means is we need to know that glutamic acid has a negatively charged R group, and aspartic acid also has a negatively charged R group when it's ionized. And so, what we're going to do is we're going to redraw our same peptide, glutamic acid, methionine, and aspartic acid over here, and we're going to consider all four of its ionizable groups. So we know that the N-terminal end of every peptide has a potentially ionizable amino group. So let's put a little blank here for the charge of this amino group. And then we know that glutamic acid has a potentially ionizable charge. And the same goes for aspartic acid's R group, could potentially have a negative charge, and the C-terminal end of every peptide could potentially have a negatively charged carboxylate anion. And so we'll leave these blank here for that. So notice that we're given this table over here with the pKa's of the amino acid residues, and we're given all of the appropriate pKa's for the four ionizable groups that we need. And so the second step in calculating the isoelectric point of a peptide is to order all of the pKa's from smallest to greatest. And so let's go ahead and do that here. So our smallest pKa is 3.5, and then, so that's our smallest one for the C-terminus, which is our carboxyl group, so let's put carboxyl above it. Then our next smallest is 3.9, and that is for our aspartic acid R group. So we can put ASP for aspartic acid's R group. Then the next smallest is going to be 4.1, which is glutamic acid's R group, so we can put 4.1 and glutamic acid above it. And then the greatest pKa is gonna be pKa of 8, which is the pKa of the N-terminal or the pKa of the amino group, so we can put 8.0 and put amino above it. So now we've completed the second step in calculating the isoelectric point of a peptide. And the third step is to just recognize that we're going to need to check any pH between the neighboring pKas and determine the predominant structure and the net charge of the predominant structure in that region. So we can pick any region that we want between any of these pKas, but let's start and pick a region, a pH within the region that's on the left since that is typically how we do things from left to right. So we can pick any pH that we want as long as it falls between these two pKas. So let's pick a pH of, let's say, 3.6. Why not? So we pick a pH of 3.6. And so, what you'll what we'll need to do is compare the pH that we've chosen to each of the individual pKas that are present. And so, let's start off by comparing the pH of the solution to the amino group pKa. And so notice that the pH of the solution, 3.6, is smaller than the pKa of the amino group. And recall from our previous lessons that when the pH is smaller than the pKa, that means that the conjugate acid predominates and the conjugate acid form of the amino group is gonna have a positive charge. It's gonna be an NH3+. So for the amino group up here, let's just put a positive charge on it so that we know that it's gonna be positively charged. So now let's move on to our next group, and our next group is glutamic acid. And so let's compare the pH to glutamic acid's, so let's compare the pH to glutamic acid's, pKa. And so the pH of 3.6 is smaller than the pKa. And again, when the pH is smaller than the pKa, the conjugate acid predominates, and the conjugate acid form of glutamic acid to R group is actually gonna have a neutral charge. And so we can go ahead and put a, 0 in here for its R group since it's gonna have a neutral charge. And, we can move on to our next group. So the pH of 3.6 is again smaller than the pKa of the aspartic acid R group. And so, again, when the pH is smaller than the pKa, the conjugate acid predominates, and the conjugate acid form of aspartic acid's R group has a neutral net charge of 0. And then for our final group, notice that the pH of 3.6 is actually greater than the pKa of the carboxyl group. And so when the pH is greater than the pKa, the conjugate base predominates. And so the conjugate base of the carboxyl group is actually going to have a negative charge, so let's put a negative charge over here. So what we're essentially saying is that within this yellow region, within any pH in this yellow region, the predominant structure of our peptide is going to have a neutral net charge since the positive charge on the amino group cancels out with the negative charge on the carboxyl group, and none of the R groups have a charge. So our peptide has a neutral net charge of 0. And when we're considering and looking for the isoelectric point, that's exactly what we're looking for, is the region that has, the predominant structure with a neutral net charge. And so what that means is all we need to do to get the pI is to take the average of the two pKas that sandwich the region that contains the neutral net charge. And so those two pKas are the pKa of the carboxyl group and the pKa of aspartic acids, R group. And so let's go ahead and calculate for the pI. So the pI or the isoelectric point is gonna be the average of those two pKas, so 3.5 plus 3.9 divided by 2, and so this comes out to 7.4 divided by 2, and if you type in your calculator 7.4 divided by 2, you get an answer of 3.7. And so what that's saying is that the pI or the isoelectric point of our peptide, glutamic acid, methionine, aspartic acid, is 3.7. And so, we know that, the peptide is going to continuously travel through the isoelectric focusing gel until it reaches the position on, the gel where the pH is equal to the pI. And so, what we essentially want to do is we want to mark the position of our peptide on this gel. And so we know that our peptide is going to travel to the pH that matches the pI, so we wanna, mark the pH of 3.7. And so here we have a pH of 3, here we have a pH of 4, and so a pH of 3.7 would be somewhere in this region right below the pH of 4. And so this is the position where the pI is, the isoelectric point, and this position, marked in red, is the final position where we expect our peptide to, end up in, after isoelectric focusing is complete. And so this concludes our practice problem here, and I'll see you guys in our next video.

Here’s what students ask on this topic:

What is isoelectric focusing (IEF) and how does it work?

Isoelectric focusing (IEF) is an electrophoresis technique used to separate proteins based on their isoelectric points (pI), which is the pH at which a protein has a net charge of zero. In IEF, a stable pH gradient is established within a gel. Proteins are loaded into the gel and an electric field is applied. As proteins migrate through the gel, they encounter different pH regions. They continue to move until they reach the region where the pH equals their pI. At this point, the protein has a neutral net charge and stops moving, resulting in distinct protein bands. This allows for the determination of the pI values of the proteins.

Why do proteins stop moving in an electric field during isoelectric focusing?

Proteins stop moving in an electric field during isoelectric focusing when they reach the region of the gel where the pH equals their isoelectric point (pI). At this pH, the protein has a net charge of zero, meaning it is electrically neutral. Since proteins with a neutral net charge do not migrate in an electric field, they cease to move further. This results in the formation of distinct protein bands at their respective pI values within the gel.

How is the pH gradient established in isoelectric focusing?

In isoelectric focusing, a stable pH gradient is established within the gel by incorporating ampholytes, which are small molecules that can carry both positive and negative charges. These ampholytes distribute themselves along the gel according to their pI values when an electric field is applied, creating a continuous pH gradient. The pH is high at the top of the gel and decreases linearly towards the bottom. This gradient remains stable and immobile throughout the process, allowing proteins to migrate to their specific pI points.

What are the applications of isoelectric focusing in proteomics?

Isoelectric focusing (IEF) is widely used in proteomics for protein separation and analysis. It helps in determining the isoelectric points (pI) of proteins, which is crucial for protein characterization. IEF is also used in two-dimensional gel electrophoresis (2-DE), where it serves as the first dimension to separate proteins based on pI before further separation by molecular weight. This technique is valuable in identifying protein isoforms, studying post-translational modifications, and analyzing complex protein mixtures in various biological samples.

What factors can affect the accuracy of isoelectric focusing?

Several factors can affect the accuracy of isoelectric focusing (IEF). These include the quality and stability of the pH gradient, the purity and concentration of the protein samples, and the presence of interfering substances such as salts or detergents. Additionally, the gel composition and the uniformity of the electric field can influence the resolution of protein separation. Proper sample preparation and optimization of experimental conditions are essential to achieve accurate and reproducible results in IEF.