SDS-PAGE Strategies - Video Tutorials & Practice Problems
On a tight schedule?
Get a 10 bullets summary of the topic
1
concept
SDS-PAGE Strategies
Video duration:
6m
Play a video:
Video transcript
In this video, we're gonna talk about some strategic ways to use SDS PAGE. So as you guys already know, SDS PAGE separates proteins only based on their mass. And unlike native page, SDS PAGE actually allows for the separation of protein subunits. However, it only allows the separation of protein subunits that are not covalently linked together. And so if we're interested in separating protein subunits that are covalently linked, then we need a way to break those covalent bonds that link them together. And that's when a molecule such as beta mercaptoethanol can come into play. So recall from our previous lessons on the Anfinsen experiment and denaturation that beta mercaptoethanol can be abbreviated with betam e, and it's specifically used to cleave covalent disulfide bonds. And so, in our example below, we're gonna talk about how we can strategically use beta mercaptoethanol with SDS PAGE to obtain information about a protein structure. And also, in the example, we're going to label each of the protein bands that we see throughout our gel with the appropriate protein subunits. And so notice that on the left over here, what we have is a protein, a single protein with quaternary structure, and that's because we have these different subunits. We have subunit a, subunit b, subunit c, and subunit d. And, what you'll notice is that subunit b and subunit d associate with subunit a via only only non covalent interactions. However, subunit a and subunit c interact with each other via non covalent interactions, but also a covalent disulfide bond that's present here. And so, that's gonna be very important to keep in mind as we analyze our gels on the right. And so notice that the first gel that we have is the native page gel. And so with native page, recall that the proteins retain their native shapes, their native charges, and their native masses. And so, essentially, the protein retains all of its native properties. And so what you'll notice is that our entire is gonna remain intact, and this single protein band that we see here is, representing all of our protein subunits. So it has subunit a, subunits b, c, and subunit d. And, remember that with native page, there are three factors that influence the migration of the protein through the gel. And again, those are the shape, the mass, and the charge of the proteins, and only proteins with native charges will actually migrate through the gel with native PAGE. Now with SDS PAGE, we are adding the molecule SDS, which is sodium dodecylsulfate. And we know that SDS is a highly non polar detergent with a negative charge. And we know that SDS is going to denature all of our subunits in our protein. And so, it's important to keep in mind that SDS, even though it denatures the proteins, it does not cleave disulfide bonds. So the disulfide bond is gonna remain intact and that means that subunit a is going to remain connected to subunit c and they're going to travel together through the gel and appear as a single single band, whereas subunit b and subunit d, which do not have disulfide bonds, are going to separate and appear as their own bands because they are different sizes. So we know that with SDS PAGE, that proteins are separated only based on their size. And so because subunit a is the largest subunit and it's connected to subunit c, it's going to be the largest protein band on our sds page gel, and the largest protein band travels the slowest. So it's going to be this protein band that's right here. And so this protein band represents sub unit a as well as subunit c because those 2 are connected via our disulfide bond that's here. So the next protein band that we see would be the next largest subunit, which is subunit b, because subunit b is greater than is larger than subunit d. So that means that the next one is gonna be subunit b, and our smallest protein band at the very bottom that travels the fastest is gonna be our smallest subunit, which is subunit d. So now, if we're interested in strategically using beta mercaptoethanol with SDS PAGE, what that means is that the beta mercaptoethanol is actually going to cleave this disulfide bond, which means that once this disulfide bond has been cleaved in the presence of s d s, these 2 subunits are finally gonna be separated from one another. And so, what you'll notice is that the largest subunit is going to travel the slowest, and so the largest subunit here would be, the one that travels the slowest, and that's going to be subunit a. Then the next largest subunit would be the one that would be next in line, and that would be subunit b, which notice, stays in the same exact position. So, subunit b has not changed its position in this gel. And so now we have an additional band here that wasn't present and this is the band for subunit c since that's the next largest behind, subunit b. So subunit c is next, and then of course the smallest subunit is going to be subunit d here, which is, which also has not changed. And so what you can see here is that, this subunit a up here at the top, it's split and it formed these two bands that we see here. And that's what these dotted red lines represent, is that this, protein band that we see here is actually representing subunit a and subunit c that were covalently bound by a disulfide bond previous to breaking it with beta mercaptoethanol. And so we'll be able to get some more practice utilizing SDS PAGE with, molecules such as beta mercaptoethanol
2
Problem
Problem
Compare the Native & SDS PAGE gels to indicate if each sample is a monomer, dimer, trimer or tetramer.
a. Sample 1: ________________
b. Sample 2: ________________
c. Sample 3: ________________
d. Sample 4: ________________
Video duration:
3m
Play a video:
Was this helpful?
3
Problem
Problem
'Protein X' has a molecular mass of 400 kDa when measured by size-exclusion chromatography. When subjected to SDS-PAGE, Protein X gives 3 bands with molecular masses of 180, 160, & 60 kDa. When SDS-PAGE is conducted a second time but in the presence of β-mercaptoethanol (β-ME), 3 bands form again, but this time with molecular masses of 160, 90, and 60 kDa. What is the subunit composition of Protein X? (Hint: draw both SDS-PAGE gels).
A
Protein X has 3 subunits with masses of 160, 90 & 60 kDa.
B
Protein X has 4 subunits with masses of 160, 90, 90 & 60 kDa.
C
Protein X has 3 subunits with masses of 180, 160, & 60 kDa.
D
Protein X has 4 subunits with masses of 180, 160, 90 & 60 kDa.