SDS-PAGE - Online Tutor, Practice Problems & Exam Prep

SDS PAGE, or Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, is a technique for protein separation based solely on mass. SDS denatures proteins, neutralizing their native charges and allowing for uniform migration through the gel. Larger proteins migrate slower, while smaller proteins move faster. By comparing unknown proteins to a ladder of known sizes, their molecular weights can be approximated. This method is crucial for visualizing protein purification, revealing the effectiveness of techniques like salting out and ion-exchange chromatography, and demonstrating the separation of quaternary structures without cleaving disulfide bonds.

SDS-PAGE

In this video, we're going to talk about SDS PAGE. So SDS PAGE is really just an acronym for a protein separation technique. And the SDS stands for Sodium Dodecyl 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 polyacrylamide gel electrophoresis. And, again, polyacrylamide is just the name of the organic compound that makes up the gel matrix. And so SDS PAGE is a protein separation technique that separates proteins only based on the mass of the protein. And so there's 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 protein that influences the migration. And so if you're wondering what sodium dodecyl sulfate is or SDS, it's just a highly nonpolar detergent that has a negative charge and it's used to denature proteins. So SDS will denature the proteins and give all of the proteins a net negative charge, and we'll talk more about how SDS works in some of our later videos. Now as we already mentioned, polyacrylamide is just the name of the organic compound that makes up the gel matrix, and polyacrylamide gel matrices are commonly used to separate proteins because they're 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 SDS PAGE, that allows us to use ladders or markers. And a ladder or marker is really just a control reference proteins of known molecular size and quantity. And so we're actually able to approximate an unknown protein's size and quantity 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, we're 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 able to work is because when we plot the log of the molecular weight versus the relative migration of the proteins in the SDS 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 2 different lanes, lane number 1 and lane number 2. And notice that in lane number 1, we have all of these different protein bands because this is referring to our 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 bands is indicated by a particular molecular size in grams per mole. And so it's the larger proteins with a larger molecular weight that are gonna travel slower through the gel. And notice that they started up here in the top of the lane and they're moving down towards the bottom of the gel, and they didn't move very far because of how large they are. But the smaller proteins, on the other hand, they move through the gel very quickly. So they started at the same position, but they moved through the gel 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 SDS 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 SDS actually works in some of our later videos. Now in lane number 2, notice what we have is our unknown protein. The protein alongside a ladder, what we're able to do is compare the migration of the unknown protein through the gel with the migration of known proteins through the gel. And so what you can see is that, the unknown protein here is migrating at a position that's right in between 45,000 and 31,000 protein markers. And so what that's saying is that our unknown protein must have a molecular size that's right about in between these two. And so if you, take the midpoint of 45,031,000 or the average of 45,031,000, so that would be add 45,000 to 31,000 divided by 2, you'll get an unknown, you'll get a molecular weight of about 38,000, and that would be grams per mole. Now, this is a more visual way to be able to approximate the mass of an 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 to plot the molecular weight, the log of the molecular weight versus the relative migration, of the proteins through the SDS gel. So, essentially, what we have over here on the right is the log of the molecular weight logMw 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 migrated through the gel, and that's what we're putting on the x axis. It's essentially, the distance, the relative migration through the gel. And on the y axis, we're putting the known molecular weights of all of these ladders. So each of these black points here represents a point on one of these protein ladders. And so notice that 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 close line of best fit. And we have we know that the formula for a line is y equals mx 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 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, you could just try to 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 SDS PAGE, and in our next lesson video, we'll able to get a better understanding of how the SDS actually works to 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:

• A. Determine a protein's isoelectric point.
• B. Determine the amino acid composition of the protein.
• C. Separate proteins exclusively based on molecular weight.
• D. Preserve a protein's native structure and biological activity.

SDS-PAGE

So now that we know that SDS PAGE is a protein separation technique that separates proteins almost exclusively on their mass, let's talk about how SDS actually works. And so the way the SDS works is that it binds to proteins approximately proportional to the molecular weight of a protein. And so there's about 1 SDS molecule that binds per amino acid residue. And so if a protein has 200 amino acid residues, there's going to be approximately 200 SDS molecules that bind to that protein. And so previously, we said that SDS is a highly nonpolar, negatively charged detergent. And so the nonpolar, negatively charged SDS will denature proteins, and the way that it denatures proteins is because of the nonpolar portion, which disrupts the hydrophobic interactions that stabilize the protein's core. And the negatively charged portion of the SDS will overwhelm and neutralize any of the native charges that are 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 to 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 SDS, this means that the native charges of a protein are no longer a factor to influence the migration of the protein through the gel. And so the only 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 SDS PAGE separates proteins exclusively on their mass. And so let's take a look at our example down below of SDS. And notice on the left here, we have an image of the sodium dodecyl sulfate structure or the SDS structure. And so notice what we have is a long hydrocarbon chain here, and we know that hydrocarbons are highly nonpolar, and that's what makes SDS a highly nonpolar molecule. And really, it's this nonpolar portion here that disrupts the hydrophobic interactions that stabilize a protein's core and denatures 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 SDS 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 going to 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 are forming, and so all of that is stabilizing the native shape of the protein. And this is before SDS treatment. Now after we treat the protein with SDS, notice what we get is our denatured protein. And so our denatured protein loses its shape. Notice that its shape is an unfolded shape now. And so, you can see that we have an 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 SDS molecule binds per amino acid residue. And so SDS allows proteins to be separated only 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 SDS also denatures quaternary structure. And recall that quaternary structure is when a protein has multiple polypeptide chains called subunits. Now, notice that down below in our example, we only have 1 polypeptide chain, so there's not any quaternary structure. But if we were to imagine a second polypeptide chain over here, which is a smaller polypeptide chain and a smaller subunit, SDS will disrupt and denature the quaternary structure. So this subunit would also be denatured. And so it would also have its own negative charges that are found on it. And because SDS PAGE separates proteins based on their size, and these 2 are different sizes, then that means that these 2 subunits would actually be separated by SDS PAGE. And because they're separated, they're going to show up and appear on the SDS 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 subunits are not separated. But with SDS, subunits can be separated. However, it's very important to keep in mind that SDS does not cleave disulfide bonds. And so, disulfide bonds are covalent linkages. And so, if these 2 subunits were actually linked via a disulfide bond, like this red bond here, the disulfide bond would not be cleaved. And so what that means is that these 2 subunits would actually be forced to migrate together through the gel and they would appear as a single band on the gel because those subunits 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 SDS can disrupt quaternary structure, but it does not cleave disulfide bonds. And so this concludes our lesson on how SDS actually works and we'll be able to get a little bit more practice in our next couple of videos. So, I'll see you there.

True or false: Protein subunits linked via disulfide bonds appear as separate bands on an SDS-PAGE gel.

• A. True.
• B. False.

SDS-PAGE

So at this point, we understand a little bit about how SDS actually works to allow for SDS PAGE to separate proteins only based on their mass. And so in this video, we're going to talk about how SDS PAGE can be implemented into our protein purification strategy. One of the main takeaways that I want you to know from this video is that SDS PAGE allows biochemists to visualize protein purification. Unlike chromatography, SDS PAGE allows both the numbers and the quantities of proteins to be visualized on a gel. Looking at our example below, we're going to see how SDS PAGE can be used to visualize the effectiveness of protein purification techniques. Notice what we have down below is an SDS 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. SDS will make all of our proteins negative, and so our proteins are going to start at the top of our gel and they're all going to migrate towards their opposite charge, so all of our proteins are migrating towards the bottom of the gel.

You'll notice that what we have are these 6 different lanes, and each lane has different contents that are labeled at the top of the lane. In the first lane, what we have is our ladder, and our ladder is in units of grams per mole. SDS PAGE separates proteins based on their mass. The larger proteins with the larger masses move slower through the gel, so they end up towards the top of the gel. But the smaller proteins with smaller masses, they move much faster to the bottom and they end up towards the bottom of the gel. In our first lane, we have the ladder and you can see how we have all of the molecular markers that correspond with these particular bands that are in the ladder.

With our crude extract, present in lane number 2, recall that the crude extract results from protein extraction, and it is a big mixture of all of the contents of the cell. It's no surprise that we have a bunch of different types of proteins. Each of these bands present in this lane represents different proteins. The intensity of the bands or the thickness of the bands tells you the quantity of that particular protein. At this point here, we have a high quantity of this particular protein. This protein up here, we have a smaller quantity of that protein.

In our 3rd lane, what we have is the same sample, but after the process of salting out. Salting out separates proteins based on their solubilities when we add salt to the solution to precipitate specific proteins that have similar solubilities. Salting out does not perfectly purify a protein, and that's exactly what we are able to visualize on the SDS PAGE gel, visualizing the effectiveness of the protein purification technique. The process of salting out in lane number 3 here does not perfectly purify our protein. 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 more protein purification techniques. We were able to visualize the effectiveness just by using SDS PAGE. That's one of the advantages of SDS PAGE.

In lane number 4, what we have is the same sample, but after ion exchange chromatography. Ion exchange chromatography is pretty effective. We were able to isolate the protein of interest, which seems to be this band right here. You can see that there are still a little bit of smudges that are present here. 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 after affinity chromatography. Affinity chromatography is one of our more effective types of chromatography. You can see that our protein band here is pretty much purified and we don't really see any other protein bands. In comparison to lane number 6, which is our purified protein control, this is a control protein that we know has been purified, it's been confirmed. You can see that after affinity chromatography, our protein of interest pretty much matches our protein control.

This shows you that our protein is purified and we're able to visualize that our protein is purified through using SDS PAGE. Again, that is one of the main takeaways that I want you to know: visualization is one of the main benefits and advantages of SDS PAGE. This concludes our lesson here of visualizing protein purification on SDS PAGE gels, and I will 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.

• A. i, ii, iii.
• B. i, ii.
• C. ii, iii.
• D. i, iii.
• E. ii.

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 2^nd 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?

Lane #5:

Lane #7:

Lane #8:

Alright. So this practice problem really makes us feel like real biochemists because we have to analyze the results of 2 different SDS-PAGE gels shown below. It says, suppose you purify a protein from liver cells, and then after different protein purification steps, you run SDS-PAGE, and the results are shown below. You can see in this first SDS-PAGE gel on the left, we have the protein ladder on the left-hand column, where we have the larger proteins that travel slower through the gel and the smaller proteins that travel faster through the gel. Secondly, we have the crude extract, which is just a big mixture of all the proteins, so it's no surprise that we have all these different protein bands in here. We want to purify our protein, so we start to perform protein purification techniques, such as salting out. After performing salting out, notice that we've gotten rid of a significant amount of proteins in comparison to the crude extract. But you'll also see that there are still proteins that are very large and proteins that are very small. All of these proteins here should have similar solubilities, since salting out separates proteins based on their solubilities. Now, gel filtration chromatography, also known as size exclusion chromatography, separates proteins based on size, and we've gotten rid of all of the large proteins and all these smaller proteins. Now, we only have proteins of similar molecular size. Now, affinity chromatography separates proteins based on their binding affinities. Looking at this lane right here, because it has one protein band, it appears that we have a purified protein. So because the problem is asking us to draw conclusions about lanes number 5, 7, and 8, here we have lane number 5. And so, it appears to have a purified protein. That's something that we can write down as a conclusion. Essentially what we'll have to take into account is that the problem continues to tell us that it runs a second SDS-PAGE gel. It takes the affinity purified sample. It takes this protein band and extracts that protein band from the gel, and it runs these proteins through a cation exchange column. The second SDS-PAGE gel shows the results for the flow through and the eluate from the cation exchange column. You'll notice in the second SDS-PAGE gel over here, we have the protein ladder in the 6th lane, or essentially, this first lane for the SDS-PAGE gel. And in the 7th lane, we have the cation column flow through. In this last lane over here, we have the cation column salt eluate. Just to refresh your memory on how cation exchange chromatography works, over here on the right, we know that cation exchange chromatography uses negatively charged stationary resin to collect and purify positively charged proteins. Positively charged proteins are going to interact with the negatively charged stationary resin, and they're going to remain essentially stuck inside the column because they're going to move slowly. Now the flow through is essentially referring to the content that's literally just going to flow straight through pretty rapidly. That's going to be the proteins that are negatively charged because these proteins here are not going to interact with the negatively charged stationary resin. Instead, they're going to repel. So in this first, in this 7th lane here with the flow through, the band that we see is going to be associated with negatively charged proteins. So we can put a negative symbol above it. These positively charged proteins that remain stuck inside the cation exchange column, we can get them to elute from the column by adding a salt. With this salt eluate here in lane number 8, we expect these to be positively charged proteins. Essentially, when we take this affinity purified sample and run it through cation exchange chromatography, we end up getting 2 different protein bands. We get a negatively charged protein band and a positively charged protein band. So that must mean that this protein band, although it appears to be a purified protein, is actually a mixture of positively and negatively charged proteins. This mixture of positively and negatively charged proteins, they have the same molecular weight because they appear at the same position on the gel. They also have similar binding affinities since they were both present and extracted with the affinity chromatography. So that's a conclusion that we can make from lane number 5. And so in lane number 7, essentially what we can say is that lane number 7 has the negatively charged proteins from lane 5. And really, that's the conclusion that we can make about lane 7. And then, of course, in lane number 8, we can say that it has the positively charged proteins from lane number 5. Essentially, what you can see here is that although proteins may appear to be completely purified with a single band. If we use other protein purification techniques, we can see that proteins that appear to be purified may not actually be purified, and there might be multiple proteins that are found within a single protein band. So that's a challenge that biochemists have to face in reality. And so that's one of the major takeaways you can take from this problem. So that concludes this problem, and I'll see you guys in our next video.

Here’s what students ask on this topic:

What is SDS-PAGE and how does it work?

SDS-PAGE, or Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, is a technique used to separate proteins based on their mass. SDS, a negatively charged detergent, denatures proteins and gives them a uniform negative charge. This ensures that the proteins' migration through the polyacrylamide gel is influenced solely by their size. Larger proteins migrate slower, while smaller proteins move faster. By comparing the migration of unknown proteins to a ladder of known molecular weights, their sizes can be approximated. This method is essential for visualizing protein purification and analyzing protein mixtures.

How does SDS denature proteins in SDS-PAGE?

SDS, or Sodium Dodecyl Sulfate, denatures proteins by disrupting their hydrophobic interactions and neutralizing their native charges. The nonpolar tail of SDS interacts with the hydrophobic core of proteins, causing them to unfold. The negatively charged sulfate group of SDS overwhelms the native charges of the proteins, giving them a uniform negative charge. This results in proteins having similar unfolded shapes and charge-to-mass ratios, allowing them to be separated based solely on their mass during SDS-PAGE.

What is the role of the ladder in SDS-PAGE?

The ladder in SDS-PAGE serves as a reference for determining the molecular weights of unknown proteins. It consists of proteins of known sizes and quantities. By comparing the migration distances of unknown proteins to those in the ladder, researchers can approximate the molecular weights of the unknown proteins. This comparison is possible because the relationship between the log of the molecular weight and the relative migration distance in the gel is linear.

How does SDS-PAGE help in protein purification?

SDS-PAGE helps in protein purification by allowing biochemists to visualize the number and quantity of proteins in a sample. By running samples through the gel at different stages of purification, researchers can assess the effectiveness of each purification step. For example, after techniques like salting out or ion-exchange chromatography, SDS-PAGE can show whether the target protein has been isolated or if further purification is needed. The intensity and position of protein bands on the gel provide insights into the purity and quantity of the proteins present.

Why doesn't SDS cleave disulfide bonds in proteins?

SDS, or Sodium Dodecyl Sulfate, is a detergent that denatures proteins by disrupting hydrophobic interactions and neutralizing native charges. However, SDS does not cleave disulfide bonds because these bonds are covalent linkages, which are much stronger than the non-covalent interactions SDS disrupts. To break disulfide bonds, reducing agents like β-mercaptoethanol or dithiothreitol (DTT) are required. Without these agents, proteins with disulfide bonds will migrate together through the gel, appearing as a single band in SDS-PAGE.