and this video, we're gonna talk about some strategic ways to use SDS page. So as you guys already know, STS page separates proteins Onley based on their mass. And unlike native Page, STS Page actually allows for the separation of protein subunits. However, it on Lea allows the separation of protein sub units that are not co violently linked together. And so if we're interested in separating protein sub units that are co violently linked, then we need a way to break those Covalin bonds that link them together. And that's when a molecule such as bottom or Capito ethanol can come into play. So recall from our previous lessons on the infants and Experiment and D nature ation that Bottom or Capito ethanol can be abbreviated with beta m E. And it's specifically used to cleave Co Vaillant Die Sulfide Bonds. And so, in our example below, we're going to talk about how we can strategically use bottom or capital ethanol with STS Page to obtain information about a protein structure, and also in the example we're going toe 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 sub units. We have sub unit, a sub unit B subunits C and sub unit deep, and what you'll notice is that sub unit B and sub unit D associate with subunits Avia Onley non Covalin interactions. However, sub unit A and sub units see interact with each other via non Covalin interactions. But also a co Vaillant Die sulfide bond that's present here. And so uh, that's going to 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 retained 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 protein is gonna remain intact. And this single protein band that we see here is representing all of our protein subunits. So it has subunits a subunits BC and sub unit deep 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 Onley proteins with native charges will actually migrate through the gel with native page. Now, with STS page, we are adding the molecule S D s, which is sodium Dodik all sulfate. And we know that S D S is a highly non polar detergent with a negative charge. And we know the STS is going to de nature all of our sub units in our protein And so it's important to keep in mind that s DS. Even though it d nature's the proteins, it does not cleave die sulfide bond. So the dye sulfide bond is going to remain intact. And that means that sub unit A is going to remain connected to sub unit C and they're going to travel together through the gel and appear as a single band, whereas sub Unit B and sub unit D, which do not have die sulfide bonds are going to separate and appear as their own bands because they are different sizes. So we know that with STS Page that proteins are separated Onley based on their size. And so because Sub Unit is the largest sub unit and it's connected to subunits, see, it's going to be the largest protein band on our STS 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 subunits. See, because those two are connected via our die sulfide bond that's here. So the next protein band that we see would be the next largest sub unit, which is sub unit B because sub unit B is greater than is larger than sub unit deep. So that means that the next one is gonna be sub unit B. And our smallest protein band at the very bottom that travels the fastest is gonna be our smallest sub unit, which is sub unit deep. So now if we're interested and strategically using bottom or capital ethanol with STS page, what that means is that the bottom or capital ethanol is actually going to cleave this die sulfide bond, which means that once this die sulfide bond has been cleaved and the presence of STS, these two sub units are finally gonna be separated from one another. And so which will notice is that the largest sub unit is going to travel the slowest. And so the largest sub unit here would be the one that travels the slowest. And that's going to be sub unit A. Then the next largest sub unit would be the one that would be next in line, and that would be sub unit B, which noticed stays in the same exact position. So Sub unit 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 Sub unit. See, since that's the next largest behind uh, sub unit B. So subunits C is next. And then, of course, the smallest sub unit is gonna be sub unit D here, which is which also has not changed. And so what you can see here is that, uh, this sub unit a appear in 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 sub Unit A and sub unit C that were co violently bound by a die sulfide bond previous to breaking it with bottom or capital ethanol. And so we'll be able to get some more practice utilizing STS page with molecules such as beta more capital ethanol in our practice problems. So I'll see you guys in those videos.
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: ________________
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“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).
Protein X has 3 subunits with masses of 160, 90 & 60 kDa.
Protein X has 4 subunits with masses of 160, 90, 90 & 60 kDa.
Protein X has 3 subunits with masses of 180, 160, & 60 kDa.
Protein X has 4 subunits with masses of 180, 160, 90 & 60 kDa.