Monday, Monday, I have a song I'm writing

called Hyundai, Hyundai, I'm sure you can figure out

the tune it goes to.

Hope everybody had a good weekend.

Anyone remember it?

Alright, enough silliness.

We spent some time last time talking about techniques

and today I'm going to spend some additional time

talking about some new techniques,

some of which I think you'll find very medically relevant.

So, hopefully, you'll find this interesting what I have to say.

Last time I was talking I finished up

talking about polyacrylamide gel electrophoresis

and more specifically, I talked about SDS-PAGE and SDS-PAGE

is what we use to separate proteins by electrophoresis

and those proteins we have to manipulate

to make them behave like DNA molecules so we manipulate them

so they behave like DNA molecules and the manipulation

that we did was that we treated them with SDS.

The SDS coated those proteins and it made them lay out flat

like rods and the coating gave them a very negative charge

and the longer that rod was,

the more negative charge it would have.

The common question I get, and I got last time was,

"Well does that negative charge negate any other charge

"that's in that protein?"

And the answer is yes it does because there's a lot more of those SDS's

on there than there are charged residues within that protein.

So that effectively makes that a polyanionic substance

meaning there are multiple negative charges.

And it's not a perfect technique.

Some proteins behave a little anomalously but the vast majority

of proteins behave very well in SDS

and we can see their molecular sizes.

The next figure I want to show you is an example of a protein gel

where on both sides of this is called a ladder

and a ladder simply is a set of proteins of known size

that are there and then you can see a protein being purified

at various steps in the purification process

and there are various quantities of that protein

being present and we can see an estimated size from that.

As I noted last time, this technique really isn't useful

for isolating a protein that we want to use later,

it's more of an analytical technique that tells us

how much protein do I have here, what's its size

and how pure is it compared to the things

I'm trying to purify it away from.

That's what SDS-PAGE is very, very useful for.

SDS-PAGE as we shall see is very useful when combined

with another technique that I'm just getting ready

to talk to you about and so I'll do that in just a moment

but before I do that I'll remind you of the structure of SDS

and no you don't have to draw SDS but this is what it looks like.

It's a long chain fatty acid and these are all carbons

and hydrogens down here and at the end we've got a sulfate

and the sulfate is what gives it

the negative charge that is necessary for electrophoresis.

Well, as I said, if we combine SDS-PAGE with another technique,

we get a really, really powerful technique

and it's a powerful technique for studying things like cancer,

for studying how drugs work and so forth, so I want to spend

a few minutes talking about that because I think

it's a pretty cool and fascinating technique.

The technique I want to discuss first is called isoelectric focusing.

Isoelectric focusing and this technique uses some substances

that are interesting in their structure,

we don't have to talk really too much about them

but suffice it to say that it relies on a mixture of compounds

that have varying amounts of charge.

Some of them are very, very, very negative

and some of them are very, very, very positive

and some of them are in between, so we can imagine,

for example, that we might have a compound that might

have forty negative charges and another compound

in our mixture that might have thirty-nine, thirty-eight,

thirty-seven, thirty-six negative charges and on the other side,

we might have something that has forty positive charges

and thirty-nine, thirty-eight, thirty-seven.

And so, we've got a complete spectrum of charges

that are in this mixture that I have.

Well if I take this mixture and I treat it very carefully,

and I put it into a tube kind of like the columns

that we've been talking about and apply an electrical field

to that tube what will happen is that the things

that are the most negatively charged will move toward

the positive electrode and the things

that are the most positively charge

will move to the negative electrode.

And if I look at that tube when I've finished

doing this electrical treatment of the tube

what I'll see is that the things that had forty minus

will be closest to the end followed by the thirty-nine minus

and the thirty-eight minus,

the thirty-seven minus, etcetera, etcetera.

And in the very middle that's where I'll see things

that had a zero charge.

Make sense?

Okay.

So this tube turns out to be really nice

because if I take this tube and instead of just putting

these compounds in there

what if I put my mixture of proteins in there as well.

Will they separate according to those charges that are in there?

And the answer is yes they will.

The proteins that are the most negatively charged

will be the closest to the positive electrode and those

that are the most positively charged

will be closest to the negative electrode.

And I'll see a spectrum of proteins across that range.

Make sense?

Now these tubes have a sort of gelatinous-like material

in them so what I've done is I've separated

these guys on the basis of charge

and in essence what I've done is I've separated

them on the basis of their pI's.

We don't have to worry about that too much at the moment,

but that's basically what I've done.

Those that have the lowest pI's are on one side

and those with the highest pI's are on the other side.

All right, now, why do I tell you that?

I tell you that because first of all that technique

is a way of separating things by their pI's

but more importantly when I combine that technique with SDS-PAGE,

I get something really, really cool.

So let's imagine we do what I just said, we take that tube,

we take our proteins, we mix them we run that electrical current

so we separate those guys on the basis of their pI

with those with the highest pIs on one side

and those with the lowest pIs on the other side

and the others, a mixture in between.

Now what if we carefully take that tube

and I open the tube up and I take that gelatinous material out

and I lay it on top of a gel where I'm going to do SDS-PAGE?

So I add SDS this material that's in here, I coat it all with negative charges,

It's not starting out with negative charges

because I haven't put anything on it to start with,

I just have the proteins by themselves, I add the SDS to it

and then I run the material on that tube downwards

and separate on the basis of size.

What I've just done is something we describe as 2-dimensional gel

electrophoresis, two dimensions.

The first dimension separated on the basis of pI

and the second dimension separated on the basis of size.

This figure schematically shows you what I just described.

Here are some proteins that have been separated

on the basis of their pI on the top and then we've added SDS

and applied them to an SDS-PAGE gel

and separated them on the basis of,

first on the basis of pI, secondly on the basis of their size.

All right, so what do we see?

We see this gel if we were to look at the upper left

we would see the proteins up in here we would see the proteins

that have the most negative charge

and are the largest.

Down here we would see the ones that have the most negative charge

and are the smallest,

down here the most neutral charge and the smallest, et cetera.

You get the idea as we go across this two dimensional gel.

Well, this turns out to be an extraordinarily powerful

analytical tool as well.

Let me show you what an actual 2-D gel looks like.

An actual 2-D gel looks something like this.

Wow what a mess, right, what a mess this thing is.

Well it turns out this mess is pretty darn cool

because this mess in this case was taken from all

of the proteins in given cell.

I can separate all the proteins

and I can decide using a variety of techniques

which protein corresponds to which spot.

Every protein will have its own unique spot.

Right, that's one thing and second, this type of analysis

if you're very, very careful is very reproducible.

It's very reproducible.

So what does this mean?

Well, let's imagine I want to study a certain cancer

and I want to understand something about that cancer.

I might take and let's say I've got liver cancer.

I might take some cells from my liver that are not cancerous

and I might separate those proteins on a 2-D gel

and then I might take that liver tumor that I have

and I might take those proteins and apply them to a different 2-D gel

and then I would compare the 2-D gels and say which proteins are different?

which proteins are being made in the cancer

but not being made in the regular cell,

which proteins are being made in the regular cell

that are not being made in the cancer cell,

which proteins are being made more in one than the other

or less than one in the other, all kinds of things I can ask

but now as the result of this analysis I can understand

the complete picture of every protein

in a normal cell versus a cancer cell.

I'm not restricted to cancer.

I could say what happens I've got this new drug

that I want to treat people with and I want to understand

what affect this drug has on our cells.

I take one batch of cells over here, don't treat it with the drug

another batch of cells over here,

treat it with the drug, do 2-D gel electrophoresis

and then ask the question, which proteins are different?

That's a really powerful tool, an extraordinarily powerful tool

This type of analysis tells us very quickly

how we're affecting the expression of proteins inside its cells.

Since proteins are the workhorses of cells,

we know very, very quickly something about the cancer,

something about the treatment,

something about your cells versus my cells.

Maybe I'm making more of one protein than your cells are.

You get the idea.

2-D gel electrophoresis is a very powerful tool.

Questions on that, yes up in the top.

Very, very good question.

His question is, if I take a protein that has quaternary structure

and I treat it with SDS, will the subunits come apart.

Let's take a vote, how many people think they will come apart?

How many people think they won't come apart?

Majority rules, the subunits will come apart and there's two reasons.

One, hydrophobic interactions can in some cases help stabilize

a quaternary structure, but even if the quaternary structure

is not being stabilized by those interactions,

when the individual subunits themselves unfold,

it disrupts completely completely the interactions

between the subunits.

So, quaternary structure is completely disrupted

by SDS treatment.

Yes?

No, actually that's not true.

What he said was, "If it denatures, then the individual strands

"are going to run faster than the entire protein."

But remember, the same happens in any kind of denaturation.

When I know I've got a multi-subunit protein,

I know what the individual subunits are.