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  • Another Monday beckons, another week beckons.

  • One day closer to an exam.

  • Student: Whoo!

  • Kevin Ahern: Yay, huh?

  • One day closer to your opportunity

  • to show me how much you know.

  • That's good.

  • I hope you had a good weekend.

  • Student: Fantastic.

  • Kevin Ahern: Fantastic?

  • Student: We won.

  • Kevin Ahern: Are we talking about football here?

  • Student: Yeah.

  • Kevin Ahern: Okay.

  • So the football team won.

  • So last time, I threw out the topic

  • to you of the 2D gel electrophoresis,

  • and I think that's a really phenomenal technology.

  • I think it allows

  • not "I think", I know it allows

  • us to do amazingly complex analyses of cells.

  • And if we have cells that have different experiences

  • one being a tumor cell, one not being a tumor cell,

  • one being treated with a drug,

  • one not being treated with a drug, one being starved,

  • the other not being starved, et cetera, et cetera

  • we we can use this technology to see

  • very clearly at the protein level

  • how these changes occur inside of the cells.

  • Several students after the class asked me if there were

  • libraries of gels that were out there that are cells

  • of known treatments.

  • The answer is, there are.

  • But many laboratories will actually do their own

  • side-by-side comparison because one of the things

  • that you see is the reproducibility is not 100% the same,

  • so if you've done both of them in your

  • laboratory at the same time,

  • you're a little bit more able to compare them.

  • So that's something that happens.

  • But, yes, there are libraries of such things out there.

  • And I just realized, I haven't checked the camera

  • to make sure it's properly on the screen.

  • So give me just a second to check that.

  • Doo-do-doo-doo.

  • And the answer is, it was perfect.

  • Alright.

  • There's nothing worse than looking at your video afterwards

  • and you see you had it about halfway on screen

  • and about halfway off the screen.

  • And you guys like that about as much as I do,

  • so, yeah, maybe less than I do.

  • One of the things I skipped over in getting to tell you

  • about 2D gel electrophoresis was to tell you about

  • gel electrophoresis itself.

  • So that's how I'm going to start the lecture today,

  • telling you how gel electrophoresis works

  • and I'm going to talk about

  • two different types of gel electrophoresis.

  • The first type I will talk about

  • is actually the simpler of the two,

  • and it is what we refer to as DNA,

  • separating DNA by agarose gel electrophoresis.

  • Agarose is, and there's the word right there

  • agarose gel electrophoresis,

  • I keep popping out here

  • agarose gel electrophoresis is a technique.

  • I don't have a figure for it anymore.

  • Your book used to have a figure and then they took that

  • away from me, so I don't have the figure out for it.

  • But I can tell you it's, in principle,

  • very much the same as polyacrylamide gel electrophoresis.

  • So let me just show you what that looks like.

  • Agarose gel electrophoresis is what we use

  • to separate fragments of DNA.

  • We can also separate fragments of RNA with it.

  • We do not use agarose gel electrophoresis to separate proteins,

  • and you'll see why that's the case in just a little bit.

  • The first reason, though, that we don't use it to separate

  • proteins is that nucleic acids are way bigger than proteins.

  • The biggest molecules in the cell are DNA molecules, by far.

  • Proteins don't even come close in terms of size.

  • What the agarose provides, in the case of DNA separations,

  • or what the polyacrylamide provides,

  • in the case of protein separations, are a matrix.

  • And we can think of this matrix

  • sort of like it's schematically shown here.

  • The matrix is a series of strands or connected

  • things that provide a support.

  • The support is to support the liquid of the buffer.

  • So just like we could take a mix of Jello

  • and put it into water and boil it,

  • when it cools down, it forms a solid support based

  • on what was in there, so, too, can we do with materials

  • for the gel, the difference being, in the case of a gel,

  • that these strands that provide the support will provide

  • little channels or little holes through

  • which the macromolecules can elute.

  • And I'll show you how that happens, okay?

  • Agarose has bigger holes than polyacrylamide does.

  • So we need those bigger holes to separate DNA molecules.

  • So how do I separate using gel electrophoresis for DNA?

  • Well, first of all, I take my DNA molecules

  • that would be a mixture of different sizes.

  • And I would apply them to the top of my gel,

  • as you can see here.

  • So I make these little indentations

  • that are what are called "wells."

  • And into these wells, we pour our mixture of DNA fragments.

  • DNA fragments are negatively charged.

  • They're polyanionic,

  • meaning that they have many, many negative charges.

  • For every base that we add, we get another negative charge.

  • So the charge is proportional to the length,

  • and the length is proportional to the length.

  • Now, you'll see why that sort of makes sense, in a second.

  • The charge is proportional to the length,

  • and the length is proportional to the length.

  • And what we do in separating these guys

  • is we use an electric field.

  • The electric field we use places a negative charge at the top.

  • You can see that little negative ion right there.

  • And it places a positive charge at the bottom.

  • The DNA molecules, being negatively charged,

  • are repelled by the negative at the top

  • and attracted toward the positive at the bottom.

  • Well since the ratio of the charge to size is constant,

  • that is the longer molecules have more charge,

  • but they also have more size

  • the separation that happens between these molecules

  • is solely on the basis of their size...

  • solely on the basis of their size.

  • The smallest guys can move the fastest through

  • these channels and they go racing through the gel.

  • The largest molecules don't have that same mobility

  • and it takes them longer to get through the gel.

  • So at the end of a stint of gel electrophoresis,

  • what we see is the gel products.

  • So this is a protein gel,

  • but a DNA gel would look very much like this,

  • where we have fragments that have been separated by size.

  • So this would be the largest molecules up here.

  • These would be the smallest molecules down here.

  • And these are specific fragments, in this case,

  • that have been purified of a protein

  • that have a given size that's there.

  • So, in principle, DNA electrophoresis and protein

  • electrophoresis are the same after we have to do some

  • manipulations to proteins to make that happen,

  • and I'll show you how that occurs.

  • So DNA electrophoresis makes sense?

  • Yes, sir?

  • Student: So if the charge on the bottom isn't

  • great enough that it's, it's not just going

  • to tear through the gel?

  • Kevin Ahern: So his question is the charge on the bottom

  • great enough that it's just going to not tear through the gel?

  • In fact the molecules will, if you leave it long enough,

  • go all the way through the gel.

  • Yes, they will.

  • So they will go all the way through,

  • this is cutting out.

  • They will go all the way through the gel.

  • So there's several variables that we have.

  • We don't need to consider them really here,

  • but I will tell you we can change the percentage of agarose,

  • which will actually change the size of those holes

  • that the DNA molecules are passing through.

  • So we can optimize that for different

  • things that we're trying to separate.

  • And I'm getting some noise.

  • Maybe that took care of it.

  • So that's DNA electrophoresis.

  • It's pretty straightforward.

  • With protein electrophoresis,

  • we've got a different consideration.

  • And the reason we've got a different consideration is,

  • first of all, proteins are globs.

  • And second of all,

  • proteins don't have a uniform mass-to-charge ratio.

  • Some proteins are going to be positively charged.

  • Some are going to be negatively charged.

  • Some are going to be neutral.

  • And that charge is really unrelated to the size of the protein.

  • So if we try to separate proteins without some other things

  • to give an artificial size-to-charge ratio that's constant,

  • then we're going to have trouble.

  • Because if I take my mixture of proteins

  • and I've got some positive ones on top

  • and some negative ones in there,

  • the positive ones aren't even going to enter the gel.

  • They're not even going to go in.

  • Boy, this is really misbehaving today.

  • Alright.

  • So I have to do something, then, to make the,

  • I have to do something to make the proteins have

  • a reasonably constant charge, or size-to-charge ratio.

  • So the trick that's used is a very clever one

  • and it works very, very well.

  • It may seem a little odd, at first, but it's actually a very,

  • very good way to give proteins an artificial

  • size-to-charge ratio that's constant.

  • What we do is take the mixture of proteins

  • that we want to separate,

  • and we add excess detergent, called SDS.

  • That stands for "sodium dodecyl sulfate."

  • So it's a long carbon chain molecule that has

  • at one end a sulfate.

  • Now, that sulfate is negatively charged.

  • When these proteins encounter the SDS,

  • if you recall when I talked about

  • what detergents can do to protein,

  • what did I say would happen?

  • They denature, they unfold.

  • So this protein that starts out as a glob,

  • first of all, elongates out into a nice long chain.

  • So, visually, we could imagine this guy is going to look

  • something like a straight DNA molecule,

  • not as big, but a straight DNA molecule.

  • The second thing that happens is these

  • sodium dodecyl sulfates completely envelope the chain.

  • Alright?

  • They just completely go all the way around the thing,

  • making like a Twinkie or something, okay?

  • A Twinkie's got the little

  • chewy center, right?

  • The chewy center being the protein,

  • and it's got this coat of stuff all the way around it.

  • Well, that coat, of course,

  • is proportional to the length of the polypeptide chain.

  • Longer polypeptide chains will have more of those

  • sodium dodecyl sulfates than smaller ones will.

  • So the size-to-charge ratio is relatively constant.

  • It's not absolutely constant,

  • but it's relatively constant.

  • And, in fact, for most purposes,

  • it's constant enough that we can get very,

  • very good separations based on size.

  • So once we've done that,

  • we take our mixture of proteins,

  • that are now all coated with this SDS,

  • and we separate them on a polyacrylamide gel.

  • And as I said earlier,

  • the only difference between agarose

  • and polyacrylamide is that polyacrylamide

  • simply makes smaller pores,

  • smaller holes, for those proteins to go through.

  • We apply an electrical current,

  • just as we did before,