Native Gel Electrophoresis - Online Tutor, Practice Problems & Exam Prep

Native gel electrophoresis, or native PAGE, separates proteins based on their native charges, shapes, and sizes using an electric field. Proteins migrate towards opposite charges, with larger proteins moving slower. The migration is influenced by mass, charge, and shape, allowing for the retention of native properties. After staining, proteins appear as bands, with thickness indicating quantity. Understanding these factors is crucial for analyzing protein behavior, as differences in charge and shape can lead to varied migration patterns, making native PAGE a valuable tool in proteomics.

Native Gel Electrophoresis

In this video, we're going to talk about native gel electrophoresis. So native gel electrophoresis is really just standard gel electrophoresis on native proteins. And recall from your previous courses that gel electrophoresis uses an electric field to separate charged molecules. So native gel electrophoresis uses an electric field to separate charged proteins based on their native charges, shapes, and sizes. All three of those factors will actually influence the migration of the protein through the gel. Native gel electrophoresis is also known as native polyacrylamide gel electrophoresis or for short, just native PAGE. The polyacrylamide is just the name of the organic compound that makes up the gel matrix for gel electrophoresis of proteins. You may already know from your previous courses that gel electrophoresis generates an electric field with a negative and a positive charge on opposite ends of the gel.

Looking down below at our example, notice that we have a power supply that allows us to generate an electric field. The negative cord here in black is hooked up to the negative electrode, which allows us to generate a negative charge on one end of the gel, and the positive cord here is hooked up to the positive electrode, which allows us to generate a positive charge on the opposite end of the gel. It's important to keep in mind that for native PAGE, it's only the proteins that have a native charge that are actually going to move and migrate through the electric field and through the gel. If the protein does not have a native charge, it will not migrate through the gel.

In this example, we also have sample wells, and this is where we're going to load our proteins into the gel. We have four different sample wells, closer to the negative electrode. It's important that proteins with a native charge are going to move towards their opposite charge through the gel. For instance, if we were to have a negatively charged protein in blue, this protein has a negative charge, it's going to move away from the negative charge and be attracted to the positive charge on the opposite end of the gel. The direction of movement for a negatively charged protein with this setup is towards the positive electrode.

If we were to have a positively charged protein with the same setup, let's put the positively charged protein in red, then this positively charged protein is going to move towards the negative electrode, and it's going to run off the gel. That is not something that you would typically want. You normally want your proteins to remain inside of the gel, so for a positively charged protein to have plenty of room to make its way towards the negative electrode and stay inside of the gel is important.

You may already know that the larger proteins are going to travel slower through the gel. If we were to have a negatively charged protein in blue that's really large, this protein would only migrate very slowly through the gel and it would end up higher in the gel, whereas a smaller protein that's negatively charged would move faster through the gel. It's also very important to keep in mind that for native gel electrophoresis, for native PAGE, the proteins are going to retain their native shapes as well as their native charges. Both the native shapes and the native charges are going to affect the gel migration as well.

After you stain the proteins, the different proteins are going to appear as different bands on the gel. The quantities of the proteins are indicated by band intensity. Essentially, after you run, you load some proteins here, negatively charged proteins, if we had a mixture of proteins, we could separate them and they would show up as bands. So you would have one band here that would represent one protein, a smaller protein would make its way further through the gel, and if it had the same intensity, that does not have to do with the size. It actually has to do with the quantity. A similar intensity, a similar thickness of the band would indicate a similar quantity of the same protein. Now if you had a smaller quantity, that would indicate less, a smaller thickness of the band, that would indicate a smaller quantity. Whereas a larger band, a thick band, would indicate a large quantity of that protein.

Over here on the right, we're going to simulate a gel electrophoresis of three different proteins, protein A, protein B, and protein C. All three of these proteins have approximately the same mass, so they are pretty much identical in mass. And what you'll notice is that protein A and protein B, not only do they have identical mass, they also have identical shapes. You can see that they have a two-subunit protein. Each of these circles represents a subunit, and they both are circles. They have the same mass and the same shape. But what you'll also notice is that they differ in their net native charge. Protein A has a net native charge of negative 5, whereas protein B has a net native charge of negative 1. Maybe because these proteins had the same mass, you might expect them to travel through the gel the same way. But with native gel electrophoresis, even though they have the same mass and the same shape, their difference in their charge actually allows them to travel differently through the gel. Notice that protein A travels farther through the gel because it has a greater net negative charge of negative 5, which means that it's going to encounter stronger forces with the electric field and be able to move further through the gel. Whereas, protein B will not travel as far through the gel because it has a smaller net negative charge.

So what that means is that also native PAGE migration in the gel is affected by the mass like you might expect, but the native charge here will also affect the migration. Now, if we compare protein B to protein C, what we'll notice is that they have the same mass and the same net charge. But it's obvious that their shapes are different from one another. Even though their masses are identical and their charges are identical because their shapes are different, notice how they migrate differently through the gel. And you can see how the shape of the protein can really have a drastic effect on the migration through the gel, which means that the shape is also going to influence the migration.

The biggest takeaway from all of this is that with native PAGE, there are three different factors that will influence the migration of the protein through the gel: the mass, the charge, and the shape. And so, there are some instances where this native PAGE would be useful because the proteins retain their native properties. They retain their native shape, they retain their native charge, and they retain their native masses. And you can see that the subunits will also stay together. So that's an important benefit. But because there are three different factors that influence the movement through the gel, it makes it a lot more difficult to be able to analyze and predict the migration of the gel of the proteins through the gel. Normally, we would only want one factor to influence the migration through the gel and that is where SDS PAGE comes into play, which might sound familiar to some of you. So we'll talk about that in our next lesson video. But first, we're going to get some practice on native PAGE. So I'll see you guys in those practice videos.

Which advantage does native gel electrophoresis provide as a protein technique?

• A. Allows separation of all native proteins as they migrate through the gel.
• B. Allows separation of all native protein subunits based on their size (large proteins travel slower).
• C. Native proteins always migrate through the electric field towards the positive end.
• D. Separates charged proteins while allowing them to retain their native conformation.

Which option below best describes the native gel electrophoresis migration for Proteins A, B, C & D (assuming equal mass & shape) considering that the buffer solution has a pH = 6.4. 

Protein A pI = 5.2, Protein B pI = 6.4, Protein C pI = 7.0, Protein D pI = 9.2

• A. A & B will migrate to the negative pole while C & D migrate to the positive pole.
• B. A will migrate to the positive pole, B will not migrate, while C & D migrate to the negative pole.
• C. A & B will migrate to the positive pole while C & D migrate to the negative pole.
• D. A will migrate to the negative pole, B will not migrate, while C & D migrate to the positive pole.

A) Consider both the peptide Gly—Pro—Ser—Glu—Thr (in a linear chain) and a cyclic peptide of the same exact sequence Gly—Pro—Ser—Glu—Thr (with a peptide bond linking the Thr & Gly). Are these peptides chemically the same? Explain.

B) Can you expect to separate the peptides above by Native-PAGE? Why or why not?

Alright. So here's a 2-part practice problem, broken up into part A above and part B below. Part A wants us to consider both the peptide—glycine, proline, serine, glutamate, and threonine in a linear chain—as well as a cyclic peptide of the same exact sequence, glycine, proline, serine, glutamate, and threonine, except with an additional peptide bond linking the threonine and glycine amino acid residues. It then asks, "Are these peptides chemically the same?" To visualize these proteins, on the left, we have the linear peptide. It turns out that most proteins in nature are actually linear, which is why we've only really talked about linear peptides. However, the cyclic peptide, although very rare, does exist, and some bacteria can create these cyclic peptides. So, it's asking us if these peptides are chemically the same. We know that with linear peptides on the N-terminal end, there's a potentially ionizable amino group, and on the opposite end, the C-terminal end, there's a potentially ionizable carboxylate group. In this cyclic peptide, notice it has this additional peptide bond between the last and the first amino acid residue. So, what happens to create this peptide bond is the carboxylate group of threonine is reacting with the amino group of glycine. We know that when peptide bonds form, they form via dehydration synthesis reactions. This arrow could represent the dehydration synthesis reaction, producing a water molecule (H2O) as a side product and the new peptide bond. Thus, the cyclic peptide is missing an H2O molecule that the linear peptide has stored in its carboxyl and amino groups. So, when it's asking us if these peptides are chemically the same, the answer is no. They are not chemically the same because they literally don't have the same atoms. The cyclic peptide is missing an H2O molecule, and it also lacks free or ionizable amino and carboxyl groups, which means it has fewer ionizable groups available. So, that's the answer for Part A, explaining if they are chemically the same. Moving on to Part B, it asks, "Can you expect to separate these peptides above by native PAGE or native polyacrylamide gel electrophoresis? Why or why not?" From our previous lessons, we know that native PAGE separates proteins based on their native properties; the proteins maintain their native shapes, masses, and charges. Clearly, looking at the cyclic peptide and the linear peptide, we can see that they definitely differ in their shape, which is already one factor that will allow us to separate these two peptides via native PAGE. So, can we expect to separate the peptides via native PAGE? The answer is yes. We can because the two peptides differ in their shape, but they don't only differ in their shape. Notice, again, that the cyclic peptide is lacking a water molecule (H2O) and, therefore, it has fewer atoms than the linear peptide, which means it has less mass. Also, the linear peptide has more ionizable groups - an amino group and a carboxylate group. Depending on the pH, they could also differ in their charges. This difference in shape, mass, and charge, which varies depending on pH, indicates that we can indeed separate them using native PAGE. This concludes the practice problem for Part B. See you guys in our next video.

Here’s what students ask on this topic:

What is native gel electrophoresis and how does it work?

Native gel electrophoresis, also known as native PAGE, is a technique used to separate proteins based on their native charges, shapes, and sizes using an electric field. In this method, proteins retain their native structures and charges, which influence their migration through the gel. The gel matrix is typically made of polyacrylamide. Proteins with a native charge will migrate towards the opposite charge; negatively charged proteins move towards the positive electrode, and positively charged proteins move towards the negative electrode. Larger proteins move slower through the gel, while smaller proteins move faster. After staining, proteins appear as bands, with the thickness of the bands indicating the quantity of the proteins.

What factors influence protein migration in native gel electrophoresis?

In native gel electrophoresis, protein migration is influenced by three main factors: mass, charge, and shape. Larger proteins migrate more slowly through the gel, while smaller proteins move faster. The native charge of the protein determines its direction of movement; negatively charged proteins move towards the positive electrode, and positively charged proteins move towards the negative electrode. Additionally, the shape of the protein affects its migration, as proteins with different shapes but the same mass and charge can migrate differently through the gel. These factors combined make native PAGE a complex but valuable tool for analyzing protein behavior.

How do you interpret the bands on a native PAGE gel?

After running a native PAGE gel and staining it, proteins appear as bands. The position of the bands indicates the relative migration of the proteins, influenced by their mass, charge, and shape. Larger proteins will be higher in the gel, while smaller proteins will be lower. The thickness of the bands indicates the quantity of the proteins; thicker bands represent a higher quantity, while thinner bands represent a lower quantity. By comparing the bands, you can infer differences in protein properties and quantities within your sample.

What are the advantages of using native gel electrophoresis?

Native gel electrophoresis offers several advantages. It allows proteins to retain their native structures, charges, and interactions, providing a more accurate representation of their natural state. This is particularly useful for studying protein complexes and interactions. Additionally, native PAGE can separate proteins based on multiple factors (mass, charge, and shape), offering a comprehensive analysis of protein properties. However, this complexity can also make it more challenging to predict migration patterns compared to other methods like SDS-PAGE, which focuses solely on mass.

How does native gel electrophoresis differ from SDS-PAGE?

Native gel electrophoresis (native PAGE) and SDS-PAGE are both techniques used to separate proteins, but they differ significantly. Native PAGE separates proteins based on their native charges, shapes, and sizes, allowing them to retain their native structures and interactions. In contrast, SDS-PAGE denatures proteins and coats them with a uniform negative charge, separating them solely based on their mass. This makes SDS-PAGE simpler to interpret, as only one factor (mass) influences migration. Native PAGE, however, provides a more comprehensive analysis of protein properties, making it valuable for studying native protein behavior and interactions.