A biochemist is attempting to separate a DNA-binding protein (...

Question

A biochemist is attempting to separate a DNA-binding protein (protein X) from other proteins in solution (proteins A, B & C). Consider the chart & answer the questions below about what type of technique is best for separation.

Table comparing proteins A, B, C, and X by isoelectric point, molar mass, and DNA binding.

A. What type of chromatography is best for separating protein X from protein A? ___________________________

B. What type of chromatography is best for separating protein X from protein B? ___________________________

C. What type of chromatography is best for separating protein X from protein C? __________________________

Accepted Answer

So this practice problem says, a biochemist is attempting to separate a DNA binding protein, protein x, from all of the other proteins in solution, which are proteins a, b, and c. Consider the chart and answer the questions below about what type of technique is best for separation. And so notice that the chart has information for proteins a, b, c, and protein x, and we're given the p I or the isoelectric point as well as the molar mass for each of the proteins, and we're also told whether or not the protein binds to DNA. And so if the protein binds to DNA, what that means is that the protein has an affinity for DNA, and that means that DNA could potentially be used as a ligand in affinity chromatography. So keep that in mind as we move along. Now part a asks, what type of chromatography is best for separating protein x from protein a? And that means that we're only gonna be focusing on protein x and protein a. And so notice looking at the isoelectric point that both protein x and protein a have about a similar isoelectric point. So, the isoelectric point of protein x is 7.8, and the isoelectric point of protein a is 7.4. And so what this means is that at about a pH of the same pH, around like 7.6 or so, both protein a and protein x are gonna have about a neutral net charge. And if they have about the same net charge, then using the charge as a means for separating them is not gonna be a good idea and ion exchange chromatography is not gonna be a good idea here. Now looking at the binding to DNA, notice that both protein a and protein x bind to DNA. And so that means that using DNA as a ligand in affinity chromatography is not gonna be a good idea to separate these two proteins. Now looking at the molar mass, notice that protein a has a molar mass of 82,000, which is almost four times larger than the mass of protein x. And so what that means is that these two proteins differ greatly in their mass and in their size. And so using size as a means, for separating these 2 proteins is gonna be a good idea since protein a is much larger than protein x. And so size exclusion chromatography is probably gonna be the best, chromatography technique to separate these 2. So down below, we can put size exclusion. Now looking at part our next part here, notice that part b asks, what type of chromatography is best for separating protein x from protein b? And again, we're only gonna be focusing on protein, b and protein x. And so notice that this time, the molar mass of protein b is about the same as the molar mass of protein x. So so that means that they're about the same size and using size exclusion chromatography this time is probably not gonna be the best idea to separate them. Now, again, looking at their DNA binding, both protein b and protein x bind to DNA. So using DNA as a ligand in affinity chromatography, again, is not gonna be a good idea to separate these two proteins. Now looking at the isoelectric point this time, notice that the isoelectric point of protein x is about double that of the isoelectric point of protein b. And so what that means is that at any pH here, protein b and protein x are gonna differ greatly in their, charges. And so using, charges as a means for separating these proteins is gonna be a good idea. And so that means that we want to use ion exchange chromatography. And so down below, we can put ion exchange chromatography. Now looking at protein, part c down below, it asks what type of chromatography is best for separating protein x from protein c? And so what that means is that we're gonna be focused on, comparing protein x and protein c. And so, this time, notice that they both have about the same isoelectric point, which means that at, a pH around the isoelectric point, they're both gonna have about a neutral net charge. And if they have the same net charge, then using the charge as a means for separating them is not gonna be a great idea. So ion exchange chromatography is not gonna be a good idea. And again, looking at their mass, notice that they have about the same mass. So that means they're they're gonna have about the same size and using size exclusion chromatography is not gonna be a good idea. But when we look at their DNA binding, capabilities, notice that protein x does bind to DNA, but protein c does not bind to DNA, which means that using DNA as a ligand in affinity chromatography is probably gonna be a good way to separate protein x from protein c. And so down below, we can put affinity for the type of chromatography that would be best for separating them. And so this concludes our practice problem, and I'll see you guys in our next video.

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