Okay, so welcome to this next video in the playlist on experimental techniques. In this video, what we're going to discuss is a technique known as sucrose density gradient high-speed centrifugation. Okay, so sucrose. density gradient high-speed centrifugation and basically this is a technique for separating out a mixture containing lots of different proteins high speed centrifugation. Okay, so in the previous video, what we did, centrifugation, that's better, centrifugation, right, so in the previous video, what we did was we, took our starting material, for instance flies, we homogenized it to get a gloop containing the protein that we want to isolate and purify and but obviously lots of other things as well such as lipids, carbohydrates.
We then centrifuged it to get a protein pellet and now what we want to do is separate out the proteins in that protein pellet and OK, so our starting thing is we have this protein pellet. OK, so what we're going to do is we're going to resuspend that protein pellet in water. So we're going to turn it into a liquid containing all of our protein from that protein pellet. OK, so we've just put it in water again. Right, now, in sucrose density gradient high-speed centrifugation, basically, you separate out the different proteins.
from one another and the way that it separates them is by their density. So the basic idea is this and you get a sucrose density gradient high-speed centrifugation column and this is like a you know it's just a glass flask basically which has sucrose in it so it contains lots of sucrose and basically the sucrose isn't just arranged randomly it's arranged in a very clever fashion. where at the top you have very low density sucrose. So the molecules are not very dense in this one.
Low density sucrose. Sucrose, does it have a double S? Sucrose, oh, does it have a, no, it doesn't have a double C.
Sucrose. I don't think it does anyway. Maybe it does have a double C.
Anyway, low density sucrose. And at the bottom, you have very high density sucrose. So.
The mass is very, very dense at the bottom and low towards the top. And then in the middle, you have all different densities of sucrose, which are gradually getting more dense as you come down, basically. So you have intermediate densities of sucrose. But the density gets bigger, gets greater as you go down the column. So intermediate density sucrose.
Let me write it with a double C and see if that looks better. Sucrose. It's one of those funny things. phenomenons isn't it when you look at a word close enough it doesn't actually look as though it's spelled right it must be one of those yeah I don't know which one it is and intermediate density sucrose but basically sucrose is this sugar that you get by combining fructose with glucose you bind fructose to glucose and I think it's just a single C so if you bind fructose to glucose you get sucrose so basically we have this this sucrose density gradient column, which has all these different densities of sucrose.
And what we're going to do is we're going to put our suspended protein at the top of this column. So here is now our protein at the top in its fluid. And then what we do is we put this whole sucrose density gradient column into a centrifuge.
So we get our centrifuge machine here, and we stick our column into that centrifuge. So initially what we have is we have this sucrose gradient, and then we have our protein sitting on the top. And what's going to happen is the centrifuge is going to spin our sucrose gradient column round and round and round and round.
And basically what happens is that the protein molecules go into this sucrose. the density of the protein molecule that determines basically where it will end up in this sucrose density gradient column. So what happens is let's say our protein liquid here contains three different types of proteins. So I'll draw it on there.
So let's say it contains Protein, which I'm going to label as the green protein here. It contains a pink protein, which I'll label as the pink protein. And it contains an orange protein. So it has these three different proteins.
And let's say the green one is the least dense. So green protein is the least dense. Then orange is the most dense.
Now, orange is the intermediate density one. And pink is the most dense. OK, so the order of density goes like so. Right. So basically, what happens when you send centrifuge this is that the protein molecules are going to go into the sucrose and they will go to the same level of uh they will they will go to the level of sucrose which has the same density as they themselves are because they will not be able to um they will not be able to go down further because the higher density sucrose molecules will basically stop them from going in they can only hope to um hope to sort of you know impede in on a sucrose there if they have an equal or greater density than it.
So basically what you find is that if we take the sucrose column out now at the end of centrifuging it, that the protein has gone into the sucrose, and that these low-density proteins will be at a certain level in this sucrose density gradient column, and these intermediate proteins will be at some intermediate level down here. And the pink protein, which was the highest density, will be at a higher a higher level down here. So basically what you have now done is you have separated your free proteins out spatially, basically.
So if you want now to just extract some of this green protein, then all you need to do is go to that layer of the sucrose column And if you take the contents from there, you end up with a lot of sucrose and protein. But sucrose and that green protein is easier to separate than the green protein and the orange protein and the pink protein altogether. OK, so what the sucrose density gradient high speed centrifugation has done is it has spatially separated the different contents of your protein. OK, by density, it separates them out by the different densities.
And load. density ones stay with the low density sucrose which is higher up this column and higher density ones go down this column basically they they go further further down and sit with the more dense sucrose Okay, and I just want to talk about how you might make a high density, a sucrose density gradient column. Well, basically, all you need to do is put lots of sucrose of different gradients into this column, and then just centrifuge it, because when you centrifuge it, things of high density go to the bottom, and the things of the low density will stay at the top.
So, what basically just centrifugation... The overall effect of centrifugation is to order things by density. So it's quite easy to make a sucrose density gradient column. And then that's what you're effectively doing when you add these proteins.
You're mixing the sucrose with proteins and again you're centrifuging it. And this centrifuge will order things according to gradient. It will order your proteins according to gradient and it will put them amongst the correct density sucrose, basically. OK, so that is one method for separating a mixture of proteins into the different components of that mixture and according to their density, basically.