5. Protein Techniques
Native Gel Electrophoresis
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concept
Native Gel Electrophoresis
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In this video, we're gonna 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 charge proteins based on their native charges, shapes and sizes. And so all three of those factors will actually influence the migration of the protein through the gel. And so native gel electrophoresis is also known as native Polly acrylamide, gel, electrophoresis or, for short, just native page. And so the poly acrylamide is just the name of the organic compound that makes up the gel matrix for ah gel electrophoresis of proteins. Now you guys may already know from your previous courses that gel electrophoresis, uh, generates an electric field with a negative and a positive charge on opposite ends of the gel. And so, looking down below at our example, notice that we have a power supply that allows us to generate an electric field, and the negative chord 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 chord years hooked up to the positive electrode, which allows us to generate a positive charge on the opposite end of the gel. And so it's important to keep in mind that for native page it's on Lee, the proteins that have a native charge that are actually going to move and migrate through the electric field and migrate through the gel. And so, if the protein does not have a native charge, it will not migrate through the gel. And so notice in this example here we also have sample wells, and this is where we're going. Toe load are proteins into the gel. And so we have here four different sample wells, and they're closer to the negative electrode. And so it's important that proteins with a native charge are gonna move towards their opposite charge through the gel. And so, for instance, if we were to have a negatively charged protein in blue, let's say this protein has a negative charge. Well, it's gonna move away from this other negative charge, is going to repel that negative charge, and it's gonna be attracted to the positive charge on the opposite end of the gel, so the negatively charged protein will move through the gel in that direction, So that would be the direction of movement for a negatively charged protein with this set up. Now, if we were toe have a positively charged protein with the same set up, let's put the positively charged protein in red. Then this positively charged proteins gonna move towards its opposite charges. Gonna move towards the negative electrode, and it's gonna run off of the gel. And 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, we would have to have a different set up where we would put our sample wells on the opposite side of the gel, and then we could load are positively charged protein, so that has plenty of room to make its way towards the negative electrode and stay inside of the gel. And so that's just something important to keep in mind about gel electrophoresis. And so you guys may already know that it's actually the larger proteins that are going to travel slower through the gel. And so if we were to have a negatively charged protein and blue. That's really large. Uh, this protein would Onley 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. But it's also very important to keep in mind that for native gel electrophoresis for native page, uh, the proteins are going to retain their native shapes as well as their native charges. And both the native shapes and the native charges are going to affect the gel migration through the gel as well. And so we'll be able to see an example of that on the right side of our example down below. Now, after you stained the proteins, they're the different proteins are going to appear as different bands on the gel, and, uh, the quantities off the proteins are indicated by the intensity and the thickness of the band. And so, essentially, after you run, um, you load some proteins in here negatively charged proteins. If we had a mixture of proteins, we could separate them and they would show up as band, so you would have one band here that represent one protein. Ah, smaller protein would make its way further through the gel. And if it had the same intensity that doesn't has nothing to do with the size. It actually has to do with the quantity. And so ah ah, 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. I mean, if you had a smaller thickness of the band, that would indicate a smaller quantity, whereas a larger band, a thick band, would indicate. Ah, large quantity of that protein. So that's another, uh, important factor to keep in mind. So over here on the right, what we're gonna do is simulate a gel electrophoresis of three different proteins protein, a protein, B and protein C. And what you'll notice is that all three of these proteins protein A, B and C they all have the approximate same mass. So they are pretty much identical in mass. And which will notice is that protein A and protein be? Not only do they have identical mass, they also have identical shapes, so you can see that they have a two sub unit protein. Each of these circles represents a sub unit, and they both are circles, so they have the same mass and the same shape, but which will also notice is that they differ in their net, uh, native Net charge. So Protein A has a native net charge of negative five, whereas protein B has a native net charge of negative one. And so maybe because these proteins had the same exact mass, you might expect them to travel through the gel the same way, but with native gel electrophoresis, even though they had the same mass and the same exact shape, their difference in their charge actually allows them to travel differently through the gel. So notice that protein A travels farther through the gel because it has a greater net negative charge of negative five, which means that it's gonna encounter stronger forces with the electric field and be able to move further through the gel, whereas protein B will not travel us far through the gel because it has a smaller net negative charge. So what that means is that it's also, uh, native page. Migration in the gel is affected by the mass like you guys might expect, but the native charge here will also affect the migration. Now, if we compare protein be tau protein, see, what will notice is that they have the same exact mass, and they have the same exact net charge. But it's obvious that their shapes are different from one another. And so even though they're 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. So the biggest take away from all of this is that with native page, it's, uh 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, uh, there are some instances where this, uh, 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. Uh, on you can see that the sub units 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 ableto analyze and predict the migration of the gel of the proteins through the gel. And normally we would Onley want one factor toe influence the migration through the gel. And that is where S D. S page comes into play. Which might sound familiar to some of you guys. So we'll talk about that in our next lesson video. But first, we're gonna get some practice on native page, so I'll see you guys in that in those practice videos.
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Problem
ProblemWhich 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.
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Problem
ProblemWhich 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.
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Problem
ProblemA) 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?
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