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

SDS PAGE is a technique that separates proteins based on mass, allowing for the analysis of protein subunits. Unlike native PAGE, SDS PAGE can separate non-covalently linked subunits, while covalently linked subunits require reduction with agents like beta mercaptoethanol to break disulfide bonds. In SDS PAGE, proteins are denatured by sodium dodecyl sulfate (SDS), which imparts a negative charge, enabling size-based separation. Understanding these interactions is crucial for studying protein structure and function, particularly in quaternary structures where subunits may be linked by both covalent and non-covalent interactions.

SDS-PAGE Strategies

In this video, we're going to talk about some strategic ways to use SDS PAGE. So as you guys already know, SDS PAGE separates proteins only based on their mass. 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. 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. That's when a molecule such as beta-mercaptoethanol can come into play. Recall from our previous lessons on the Anfinsen experiment and denaturation that beta-mercaptoethanol can be abbreviated as βME, and it's specifically used to cleave covalent disulfide bonds.

In our example below, we're going to talk about how we can strategically use beta-mercaptoethanol with SDS PAGE to obtain information about a protein structure. 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. Notice that on the left over here, what we have is a protein with a quaternary structure, because we have these different subunits: subunit a, subunit b, subunit c, and subunit d. Subunit b and subunit d associate with subunit a via 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. That's going to be very important to keep in mind as we analyze our gels on the right.

Notice that the first gel that we have is the native PAGE gel. With native PAGE, recall that the proteins retain their native shapes, their native charges, and their native masses. Essentially, the protein retains all of its native properties. What you'll notice is that our entire protein is going to remain intact, and this single protein band that we see here represents all of our protein subunits. It has subunit a, subunits b, c, and subunit d. Remember that with native PAGE, there are three factors that influence the migration of the protein through the gel. Again, those are the shape, the mass, and the charge of the proteins, and only proteins with native charges will migrate through the gel with native PAGE.

Now, with SDS PAGE, we are adding the molecule SDS, which is sodium dodecyl sulfate. We know that SDS is a highly non-polar detergent with a negative charge. SDS is going to denature all of our subunits in our protein. It's important to keep in mind that SDS, even though it denatures the proteins, does not cleave disulfide bonds. So the disulfide bond is going to remain intact, and that means that subunit a is going to remain connected to subunit c and they are going to travel together through the gel and appear as a 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. With SDS PAGE, proteins are separated only based on their size. 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.

This protein band represents subunit a as well as subunit c because those two are connected via our disulfide bond. The next protein band that we see would be the next largest subunit, which is subunit b, because subunit b is larger than subunit d. That means that the next one is going to be subunit b, and our smallest protein band at the very bottom that travels the fastest is going to be our smallest subunit, which is subunit d.

Now, if we're interested in strategically using beta-mercaptoethanol with SDS PAGE, it means that beta-mercaptoethanol is going to cleave this disulfide bond, which means that once this disulfide bond has been cleaved in the presence of SDS, these two subunits are finally going to be separated from one another. What you'll notice is that the largest subunit is going to travel the slowest, and so the largest subunit here would be subunit a. Then the next largest subunit would be the one that would be next in line, which would be subunit b, which, notice, stays in the same exact position. Subunit b has not changed its position in this gel. 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. Subunit c is next, and then, of course, the smallest subunit is going to be subunit d here, which also has not changed. What you can see here is that this subunit a at the top has split and it formed these two bands that we see here. This is what these dotted red lines represent: 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.

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: ________________

So this practice problem wants us to compare the native and SDS PAGE gels to indicate if each sample is a monomer, dimer, trimer, or tetramer. Recall that a monomer only has one polypeptide chain, dimers have two polypeptide chains, trimers have three polypeptide chains, and tetramers have four polypeptide chains. Below on the left, we have our native PAGE gel where the proteins retain their native conformations, and on the right, we have our SDS PAGE gel, where SDS denatures the proteins. Looking at sample number 1 in this lane on the native PAGE, notice that its native conformation has a mass of 180 kilodaltons. But, looking at sample number 1 in the presence of SDS where it's denatured, notice that it shows up around 60 kilodaltons. This comparison shows that our native protein of 180 kilodaltons, on the native PAGE, divided by the 60 kilodaltons seen when in the presence of SDS, tells us there are three subunits. Since there are three subunits, it is a trimer. We can thus assign trimer for part a. Moving on to part b for sample 2, under native PAGE conditions, the protein shows up around 75 kilodaltons. With sample 2 in the presence of SDS, it also shows up around 75 kilodaltons. Using the division of 75 by 75, it results in 1. This tells us that we only have one polypeptide chain and this sample is a monomer. For sample 3, under native PAGE, the native conformation shows up around a mass of about 60 kilodaltons. Meanwhile, under SDS PAGE conditions, it shows up right in between the 25 and 35 markers, which averages to about 30 kilodaltons. Dividing 60 by 30 yields 2, which tells us that it is a dimer with two subunits. Finally, looking at sample 4, in the presence of native PAGE, it shows up around a mass of about 100 kilodaltons. But in the presence of SDS, there is only one band showing up at 25 kilodaltons. Taking the mass of our native protein, which is 100 kilodaltons, and dividing it by the mass of the subunits showing up at 25 kilodaltons, tells us there are four subunits. Therefore, this sample is a tetramer. Part d thus concludes with the identification of sample 4 as a tetramer. This concludes this practice problem, and I look forward to seeing you guys in our next video.

'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.

Here’s what students ask on this topic:

What is the purpose of using SDS in SDS-PAGE?

SDS, or sodium dodecyl sulfate, is used in SDS-PAGE to denature proteins and impart a uniform negative charge. This ensures that proteins are separated based solely on their mass, rather than their shape or charge. SDS disrupts non-covalent bonds, causing proteins to unfold into linear chains. This uniform negative charge allows the proteins to migrate towards the positive electrode during electrophoresis, with smaller proteins moving faster through the gel matrix. This separation by size is crucial for analyzing protein subunits and understanding protein structure and function.

How does beta mercaptoethanol aid in SDS-PAGE analysis?

Beta mercaptoethanol is a reducing agent used in SDS-PAGE to break disulfide bonds between protein subunits. Disulfide bonds are covalent links that can hold protein subunits together. By cleaving these bonds, beta mercaptoethanol allows the individual subunits to separate and migrate independently through the gel. This is particularly useful for analyzing proteins with quaternary structures, where subunits may be linked by disulfide bonds. The separation of these subunits provides detailed information about the protein's composition and structure.

What is the difference between SDS-PAGE and native PAGE?

SDS-PAGE and native PAGE are both electrophoresis techniques used to separate proteins, but they differ in their approach. SDS-PAGE uses sodium dodecyl sulfate (SDS) to denature proteins and impart a uniform negative charge, allowing separation based solely on mass. In contrast, native PAGE maintains the proteins' native state, preserving their shape, charge, and mass. This means that in native PAGE, proteins are separated based on a combination of these factors. SDS-PAGE is useful for analyzing protein subunits, while native PAGE is better for studying protein complexes and interactions in their native form.

Why is it important to understand the migration of proteins in SDS-PAGE?

Understanding the migration of proteins in SDS-PAGE is crucial for accurately interpreting the results of the electrophoresis. Proteins are separated based on their size, with smaller proteins migrating faster through the gel. By analyzing the position of protein bands, researchers can determine the molecular weight of the proteins and identify different subunits. This information is essential for studying protein structure, function, and interactions. Additionally, knowing how proteins migrate helps in troubleshooting experimental issues and optimizing the conditions for better resolution and accuracy.

How can SDS-PAGE be used to study protein quaternary structure?

SDS-PAGE can be used to study protein quaternary structure by analyzing the individual subunits of a protein complex. In the presence of SDS, proteins are denatured and separated based on their size. If the protein subunits are linked by disulfide bonds, a reducing agent like beta mercaptoethanol can be used to break these bonds, allowing the subunits to separate and migrate independently. By comparing the migration patterns with and without the reducing agent, researchers can determine the composition and arrangement of the subunits, providing insights into the quaternary structure of the protein.