in this video, we're gonna talk about SDS page. So STS Page is really just an acronym for a protein separation technique and the S. D s stands for sodium dough Dekel sulfate and the page should sound familiar to you guys from our previous lesson on native gel electrophoresis. And that's because native gel electrophoresis is also known as native page, and the page just stands for Polly acrylamide gel, electrophoresis and again, Polly Acrylamide is just the name of the organic compound that makes up the gel matrix. And so STS Page is a protein separation technique that separates proteins Onley based on the mass of the protein. And so there is only one factor that influences the migration of the protein through the gel, and that is the mass of the protein. And that's a lot different than native page because with native page, there are three factors that influence the migration of the protein through the gel, and that is the native charge, the native shape and the native mass. But with STS page, it's on Lee, the mass of the protein that influences the migration. And so, if you're wondering what sodium dough Dekel sulfate is, or STS. It's just a highly non polar detergent that has a negative charge, and it's used to de nature proteins. So STS will de nature the proteins and give all of the proteins a net negative charge. And we'll talk more about how STDs works and some of our later videos. Now, as we already mentioned, Polly Acrylamide is just the name of the organic compound that makes up the gel matrix and Polly acrylamide. Gel matrices are commonly used to separate proteins because it's proven to be quite effective for separating proteins. Now recall from our previous lesson video that it's the larger proteins that will actually travel slower through the gel. During gel electrophoresis and the smaller proteins, they're gonna travel much faster through the gel. So keep that in mind. And because there's only one factor that influences the migration of the protein through the gel with STS page that allows us to use ladders or markers. And a lateral marker is really just a control reference proteins of known molecular size and quantity. And so we're actually able to approximate a pro an unknown proteins, size and quantity just by comparing. So just by comparisons to the ladder. So by comparing the migration of the unknown protein to the migration of proteins that are part of our ladder, were actually able to approximate the size and quantity of the unknown protein. And we'll be able to see how this works a little bit down below in our example. And so the reason that this is ableto work is because when we plot the log of the molecular weight versus the relative migration off the proteins in the STS page gel, it actually turns out to be a linear relationship. And again, we'll be able to see that down below in our example. So in our example of SDS page on the Left, what we have is an SDS page gel, and we have two different lanes Lane number one and Lane number two, and notice that in Lane number one, we have all of these different protein bands because this is referring to are ladder and our ladder has a bunch of different proteins of known molecular size and quantity, and so you can see that each of these protein band is indicated by a particular molecular size. Ingram's promote. And so it's the larger proteins with larger molecular weight that are going to travel slower through the gel and noticed that they started up here in the top of the lane and they're moving down towards the bottom of the gel, and it didn't move very far because of how large it is. But the smaller proteins, on the other hand, they moved through the gel very quickly, so they started the same position. But they moved to the jail much, much faster. And we know that gel electrophoresis generates an electric field with a negative charge on one end of the gel and a positive charge on the other end of the gel. And because STS has a negative charge on it, the proteins are all going to end up having a negative charge regardless of their native charge. And again, we'll talk more about how STS actually works, and some of our later videos, now in lane number, to notice what we have is our unknown protein. So we have no idea what the molecular sizes of this protein, but when we run STS page of the unknown protein alongside a ladder, what we're able to do is compare the migration of the unknown protein through the gel What? The migration of known proteins through the gel. And so what you can see is that, uh, the unknown protein here is migrating at a position that's right in between 45,000 and 31,000 protein markers. And so, with that saying is that are unknown. Protein must have a molecular size. That's right about in between these two. And so if you take the midpoint of 45,000 and 31,000, or the average of 76,000, so that would be add 45,000 to 31,000 divided by two, you'll get an unknown. Uh, you'll get a molecular weight of about 38,000, and that would be grams promote. Now, this is, um, or visual way to be able to approximate the mass of unknown protein so you can see how we were able to use the ladder to determine the mass of the unknown protein, which we said visually looks about 38,000. But a more accurate way to be able to approximate the mass of the unknown protein is toe plot. The molecular weight, the log of the molecular weight versus the relative migration of the proteins through the STS gel. So essentially, what we have over here on the right is the log of the molecular weight and the relative migration of the proteins to the gel on the X axis. And so we're able to measure the distance that each of these protein ladders were, uh migrated through the gel. And that's what we're putting on. The X axis is the essentially the distance. The relative migration through the gel and on the Y axis were putting the known molecular weights of all of these ladders. So each of these black point here represents a point on, uh, one of these protein ladders. And so notice that the relationship, if we were to draw a line of best fit between this point, it's between all of these points. It's really a linear relationship between the log of the molecular weight and the relative migration through the gel. And that's what we said previously. It's a linear relationship, and you can see this blue line going through here is a very a clothes line of best fit, and we have We know that the formula for a line is why equals M X plus B. And so we're able to use the formula for the line of best fit to determine the mass of the unknown protein. So what we can do is we can measure the migration of the unknown protein through the gel, and we can use the, uh, formula for the line of best fit to determine where on the line does that fall. And what MASS does that correspond with to determine the mass of the unknown protein? And that is a more accurate way to be able to approximate the mass of a protein. But also, uh, you could just try toe eyeball it, and that would probably be close enough. But it depends on what type of experiment that you're trying to perform and how much accuracy you really need. And so this concludes our lesson on STS page, and in our next lesson, video will be able to get a better understanding of how the STS actually works toe be able to get us these results. And so I'll see you guys in our next video
By adding SDS to a protein and performing gel electrophoresis, it is possible to:
Determine a protein’s isoelectric point.
Determine the amino acid composition of the protein.
Separate proteins exclusively based on molecular weight.
Preserve a protein’s native structure and biological activity.
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So now that we know the STS pages, a protein separation technique that separates proteins almost exclusively on their mass, let's talk about how STS actually works. And so the way the STS works is that it binds to proteins approximately proportional to the molecular weight of a protein. And so there's about one STS molecule that binds per amino acid residue. And so if a protein has 200 amino acid residues, there's gonna be approximately 200 STS molecules that bind to that protein. And so previously, we said, the STS is a highly non polar, negatively charged detergent, and so the non polar, negatively charged STS will de nature proteins and the way that it D nature's proteins is because of the non polar portion which disrupts the hydrophobic interactions that stabilize the proteins core. And the negatively charged portion of the S. D s will overwhelm and neutralize any of the native charges that air present on a protein. And so this results in all of the proteins having very similar unfolded shapes as well as very similar charge to mass ratios. And so because all of the proteins have very similar unfolded shapes, this means that the native shape of a protein is no longer a factor. Toe influence the migration of the protein through the gel and because the native charges of a protein are neutralized by the negative charge on the S. D. S. This means that the native charges of a protein are no longer a factor. Thio influence the migration of the protein through the gel and so the Onley factor that remains that actually does influence the migration of the protein through the gel is the mass of the protein. And that's why STS page separates proteins exclusively on their mass. And so let's take a look at our example down below of STS and notice on the left. Here we have an image of the sodium Dodik Oh sulfate structure or the S. D s structure. And so notice what we have is a long hydrocarbon chain here, and we know that hydrocarbons are highly non polar. And that's what makes STS ah, highly non polar molecule. And really, it's this non polar portion here that disrupts the hydrophobic interactions that stabilize the proteins core and D nature's the protein. Now notice that the sulfate group up at the top here has this negative charge that's associated with it. And it forms an ionic interaction with a sodium molecule. And so before STs treatment notice that what we have is a protein that has a very particular shape to it. And so it has its native shape and this is our native protein, and the native protein is gonna have native charges as well as its native shape. So you can see we have these dotted lines here that represent hydrogen bonds stabilizing its secondary structure. We have positive charges. We have negative charges. We have ionic interactions that air forming. And so all of that is stabilizing the native shape of the protein. And this is before STS treatment. Now, after we treat the protein with STS notice, what we get is our denatured protein, and so are denatured protein loses its shape, noticed that its shape is an unfolded shape now and so you can see that we haven't unfolded shape and the protein also has all of these negative charges. So you can see all of these negative charges that surround the protein and the negative charges are proportional to the mass of the protein and That's because one STS molecule binds per amino acid residue. And so STS allows proteins to be separated Onley based on their mass because the shape is no longer an influence and the charge is no longer an influence. And so the last thing I want to leave you guys off with is that STS also de nature's Quaternary structure and recall that quaternary structures when ah protein has multiple poly peptide chains called sub units. Now notice that down below. In our example, we only have one poly peptide chain, so there's not any questionnaire structure. But if we were to imagine a second poly peptide chain over here, which is a smaller poly peptide chain and a smaller sub unit, uh, STS will disrupt and d nature the question eri structure. So this sub unit would also be denatured and so it would also have its own negative charges that were found on it and because, uh, STS page separates, uh, proteins based on their size, and these two are different sizes. Then that means that these two sub units would actually be separated by STS Page, and because they're separated, they're going to show up and appear on the STS page gel as separate bands. And so that's something that's very different from native page because with native page, the protein retains its native shape and sub units are not separated. But with STS sub units can be separated. However, it's very important to keep in mind that s DS does not cleave die sulfide bonds and so die sulfide bonds are co Vaillant linkages. And so, if these two sub units were actually linked via di sulfide bond like this red bond here, the dice sulfide bond would not be cleaved. And so what that means is that these two subunits would actually be forced to migrate together through the gel, and they would appear as a single band on the gel because this sub units have not been separated. And so we'll be able to get a lot more practice with this idea as we move along through our course. But for now, I just want you guys to know that STS can disrupt Quaternary structure, but it does not cleave die sulfide bonds. And so this concludes our lesson on how STS actually works, and we'll be able to get a little bit of practice in our next couple of videos. So I'll see you guys there
True or false: Protein subunits linked via disulfide bonds appear as separate bands on an SDS-PAGE gel.
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So at this point, we understand a little bit about how STS actually works to allow for STS page two separate proteins Onley based on their mass. And so in this video, we're gonna talk about how STS page can be implemented into our protein purification strategy. And so one of the main takeaways that I want you guys to know from this video is the STS page allows biochemist toe visualize protein purification. And so, unlike chromatography, STS page allows both the numbers and the quantities of proteins to be visualized on a gel. And so, looking at our example below, we're going to see how STS page can be used to visualize the effectiveness of protein purification techniques. And so notice what we have down below is an STS page gel. And we know gel electrophoresis generates a negative charge on one end of the gel and a positive charge on the other end of the gel, and STS will make all of our proteins negative and so are proteins are going to start at the top of our gel, and they're all gonna migrate towards their opposite charge. So all of our proteins are migrating towards the bottom of the gel, and you'll notice that what we have are these six different lanes, and each lane has different contents. That air labeled at the top of the lane and notice that in the first lane over here, what we have is our ladder and our ladders and units of grams per mole. And what you'll see is that STS page separates proteins based on their mass, so the larger proteins with larger masses move slower through the gel so they end up towards the top of the Joe. But the smaller proteins with smaller masses, they move much, much faster to the bottom, and they end up towards the bottom of the gel. And so, in our first lame, we have the ladder, and you could see how we have all of the molecular markers that correspond with these particular bands that are in the latter now with our crude extract that is present in lane number to recall that the crude extract is what results from protein extraction, and that is a big mixture of all of the contents of the South. So it's no surprise that we have a bunch of different types of protein So what you'll see is that each of these bands that air present in this lane right here all of these different bands represent different proteins. And the intensity of the bands or the thickness of the band's, tells you the quantity of that particular protein. So you can see that at this point here we have a high quantity of this particular protein and this protein. Up here we have a smaller quantity of that protein, and so, uh, in our third lane, what we have is the same sample. But after the process of salting out and we know that salting out separates proteins based on their soluble, it ease when we add salt to the solution to precipitate the specific proteins that have similar soluble it ease. And so salting out, we know does not perfectly purify a protein. And that's exactly what we're able to visualize on the STS page gel. Visualize the effectiveness of the protein purification technique so we can see that the process of salting out in lane number three year does not perfectly purify our protein, so you can see that we have a bunch of different bands that are still present after the process of salting out, which means that we still have a protein mixture after the process of salting out. And we have to continue to use mawr protein purification techniques. And we were able to do visualized the effectiveness just by using STS page. And that's one of the advantages of STS page. So in Lane number four, what we have is the same sample, but after ion exchange chromatography. And so what you'll see is that ion exchange chromatography is pretty effective. We were able to isolate the protein of interest, which seems to be this band right here, and you can see that there are still a little bit of smudges that air present here, so perhaps the protein is not perfectly purified, but you can see that the effectiveness of the protein purification technique in comparison to salting out is much better. And we've isolated this protein of interest, which is this band here. Ah, little bit better. And so in Lane number five, which will see, is that we have the same sample, but after affinity chromatography, and we know that affinity chromatography is one of arm or effective types of chromatography, and so What you can see is that our protein band here is pretty much purified and we don't really see any other protein bands. And in comparison, toe Lane number six, which is our purified protein control. This is a control protein that we know has been purified. It's been confirmed, and you could see that after affinity chromatography that are protein of interest, Pretty much matches are protein control. And so this shows you that our protein, um, is purified and were ableto visual eyes that are protein is purified through using STS page. And again, that is one of the main takeaways that I want you guys to know is that, uh, the visual ization is one of the main, um, uh, benefits and advantages of STS page. And so this concludes our lesson here of visualizing protein purification on STS page gels and I'll see you guys in our next video
Which of the following statements are true regarding the treatment of proteins with SDS?
i) Only proteins with native net charges acquire an overall net negative charge.
ii) Proteins denature due to a disruption of the hydrophobic interactions stabilizing the core of their structures.
iii) All protein subunits can be separated via SDS-PAGE.
i, ii, iii.
Suppose you purify a protein from liver cells and the SDS-PAGE results after different purification steps are shown. You then take the affinity purified sample and run it through a cation exchange column. The 2nd SDS-PAGE shows the results for the flow through and eluate from the cation exchanger. Based on this data, what conclusions can you draw from the results in lanes #5, 7 & 8?