Affinity Chromatography - Video Tutorials & Practice Problems
On a tight schedule?
Get a 10 bullets summary of the topic
1
concept
Affinity Chromatography
Video duration:
6m
Play a video:
in this video, we're gonna talk about affinity chromatography, so affinity Chromatography is another type of column chromatography that purifies a protein based on its affinity and by affinity. What we really mean are the specific binding capabilities of the target protein of interest that we're trying to purify. And it turns out that affinity chromatography is one of the most effective types of chromatography that we've talked about thus far, which means that it does the best job at purifying our target protein of interest. And that's a great thing. But a negative about it is that because affinity chromatography is one of the more sophisticated types of chromatography, it also makes it one of the more expensive types of chromatography. And so the stationary phase material that's packed inside an affinity chromatography column is actually co violently linked toe what's known as a lie gand and all a lie. Gand is is just a small substance that specifically binds to a bio molecule, such as a protein to form a complex with it. And so the way that affinity chromatography works is that our target protein of interest that we're trying to purify is going toe bind to the lie again that stuck to the stationary phase, and we know that the stationary phase does not move, which means that our protein of interest is going to remain inside of the column and all of the other proteins are not going to bind to the ligand, and they're going to be washed out of the column with the mobile phase. So let's take a look at our example down below affinity chromatography to clear this up. And what you'll see is that we have a bunch of different columns here, so affinity chromatography is a type of column chromatography and packed inside of the column is our stationary phase, which we know does not move throughout our entire process and the stationary phases, this blue material that's packed inside the column. And if we zoom in on the stationary phase, what will notice is that the Stationary Phases Co. Violently linked to these Liggins, which are these y shaped structures and this image. And so the lie Gans are just small molecules that will bind to a specific biomolecules interest, which is our protein, our target protein of interest and our target protein of interest is represented by this pink protein here, so notice that are pink protein fits and binds perfectly to our ligand, which is attached to the stationary phase and does not move. And so down below. In our beaker we have this big protein mixture. And with our protein mixture, we have positively charged proteins and red. We have negatively charged proteins and blue. We have neutral charged proteins and gray, and then we have our specific target protein of interest that we're trying to purify. And that's the triangular protein that's in pink. And so if we take our protein mixture and we pour it into our affinity chromatography column, then our protein mixture is gonna be at the top of our affinity chromatography column. And inside of this container at the top of our column here is our mobile phase, and we know that we're gonna be adding our mobile phase throughout the entire process. And so, as we start to add mobile phase, which starts the happen is our proteins begin to separate, so notice that are proteins are separating and our target protein of interest the pink proteins notice that they haven't moved very much, and that's because they bind to the specific lie gains, which are stuck to the stationary face so they don't move throughout the column, whereas all of the other proteins down here notice that they're moving fairly quickly. And as we continue to ADM. Or and Mawr Mobile Phase eventually will get all of the other proteins to allude from the column and be removed from the column, whereas our target protein of interest is still bound to those Liggins in our stationary face. So our target protein of interest is essentially purified inside of the column. So now how do we get these bound target proteins out of the column? How do we dilute these target proteins from the column? And so what we can do is the bound target. Proteins can later be eluded from the affinity chromatography column by adding a soluble ligand or salt to the mobile phase. And so what you can see is up here in our image. We are adding a soluble ligand to the mobile phase, and so the soluble lie again is literally just a lie again. So it looks just like the lie Ganz that air over here, except it's a little bit different. It's It's a green shape, lying in a green colored lie again, and this leg in has a stronger attraction to the target protein. So when we put it inside the mobile phase and we run the mobile phase through the column, it's gonna grab Elin toe all of the target protein. And eventually what we're gonna get is all of our target protein alluded at the bottom, and our target protein is attached to the soluble lie again that we put into the mobile phase. And so if we wanted to remove our target protein from the lie again, then we could go ahead and add a little bit of salt thio. Decrease the strength of the interactions between the protein and the Lagan. And so there's actually an advantage for using soluble Liggins versus using salt to elude our protein. So by using a soluble lie again that has a specific binding affinity to our protein that actually adds another level of specificity to our purification process. So it helps to make sure that we're really purifying our protein because we're adding another, uh, level of specifications here by adding a lie again that's specific to our target protein of interest. And that just means that we're gonna have a, uh we have mawr, um, confidence that our protein is going to be purified, but a negative to using soluble like and is that it's going to be expensive. So, like, ants can be quite expensive. And so if we're looking for, um or, uh, cheap way or a cheaper way Thio dilute our proteins, then we could just use salt to elude our protein. And so this concludes our lesson on affinity chromatography, and we'll be able to get a lot more practice, uh, utilizing all these concepts in our practice video, so I'll see you guys there.
2
Problem
Problem
In your own words, describe the principles involved in protein purification by affinity chromatography.
Video duration:
2m
Play a video:
Was this helpful?
3
Problem
Problem
The target protein to be purified is likely eluted from the affinity chromatography column by _______________. Explain potential advantages & disadvantages of the elution strategies.
A
Altering the pH of the mobile phase.
B
Addition of a chaotropic agent such as urea.
C
Addition of salt and/or free ligand.
D
Raising the temperature in the column.
4
Problem
Problem
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.
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? __________________________
