Suppose you purify a protein from liver cells and the SDS-PAGE...

Question

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:

SDS-PAGE results showing protein purification steps from liver cells, with labeled lanes for analysis.

Lane #5:

Lane #7:

Lane #8:

Accepted Answer

Alright. So this practice problem really makes us feel like real biochemists because we have to analyze the results of 2 different s d s page gels shown below. And so 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. So 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. The second layer, we have the crude extract, which is just a big mixture of all of the proteins. So it's no surprise that we have all of these different protein bands in here. And so we want to purify our protein, so we start to perform protein purification techniques, such as salting out. And so after performing salting out, notice that we've gotten rid of a significant amount of proteins in comparison to the crude extract. But what you'll also see is that there are still proteins that are very large and proteins that are very small. And so, all of these proteins here should have similar solubilities, since salting out separates proteins based on their solubilities. Now, gel filtration chromatography is also known as size exclusion chromatography. Filtration chromatography is also known as size exclusion chromatography. So notice that it separates proteins based on size, and we've gotten rid of all of the large proteins and all of these smaller proteins. And now, we only have proteins of similar molecular size. Now, affinity chromatography separates proteins based on their binding affinities. And looking at this lane right here, because it has one protein band, it appears that we have a purified protein. And 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 what we can say is that it appears to have a purified protein. So, that's something that we can write down as a conclusion. And, 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. And essentially, what it does is it takes the affinity purified sample, so essentially, it takes this protein band and it extracts that protein band from the gel, and it runs these proteins through a cation exchange column. And so, it says that the 2nd SDS PAGE gel shows the results for the flow through and the eluate from the cation exchange column. Now, what you'll notice is in the 2nd 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. And so, in this last lane over here, we have the cation column salt, eluate. And so just to refresh your guys' memories on how cation exchange chromatography works, over here on the right, we know that cation exchange chromatography uses negatively charged stationary resin, which is what's shown here, to collect and purify positively charged proteins. So positively charged proteins are gonna interact with the negatively charged stationary resin, and they're gonna remain essentially stuck inside of the column because they're going to move really, really slowly. Now the flow through is essentially referring to the content that's literally just going to flow straight through pretty rapidly. And that's going to be the proteins that are negatively charged, because these proteins here are not gonna interact with the negatively charged stationary resident. Instead, they're gonna repel. And so that's why they, flow through the fastest. And so in this first, in this 7th lane here with the flow through, essentially, the band that we see is going to be associated with negatively charged proteins. So we can put a negative symbol above it. And then, 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. And so, with this salt eluate here in, lane number 8, we expect these to be positively charged proteins. And so essentially what we can see here is that 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, it's actually so it's actually a mixture. So actually a mixture of pot of both positively and negatively charged proteins. And so this mixture of positively and negatively charged proteins, they have the same molecular weight because notice that they appear, at the same position on the gel. So they appear at the same position on the gel. So they have the same molecular weight, and they also have similar binding affinity since they were both present and extracted with the affinity chromatography. So they have same molecular weight and binding affinities. 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, so negative 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 they it has the positively charged proteins from lane number 5. And so 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 actually, 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.

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