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,