[students cheering]

It never fails to elicit a few whoos!

By the end of the term it's going to be like, "uhh."

Now it's still the, "Whoo!"

Big weekend plans?

Studying Biochemistry, this is good.

The heck with Biochemistry, right?

Alright so we're pretty much on target with where we need to be

and I'm very happy with that,

and I'm very happy with the interactions I've had

with many of you so far so it's been very positive for me.

I very much enjoy that.

We're turning our attention now,

as I said last time, to thinking about

using our knowledge of protein structure as a way of

working with proteins.

Biochemists are pretty nerdy people.

And as I probably say further in the term

and probably next term as well,

"Biochemists are also lazy people."

So we like to find easy ways to do things,

and that's one of the things that we work on pretty hard.

You'll hear some of those

later as I talk about some of the techniques.

So we are turning our attention for a couple of lectures

to techniques that we use in Biochemistry

specifically to isolate large,

and in some cases small molecules.

It's mostly relevant for the proteins we've been talking about

but we'll talk about some other things as well.

The number one tool you see biochemists use in a laboratory,

you can't walk into a biochemistry laboratory

without finding a centrifuge.

So a centrifuge is something that is ubiquitous

and it's a tool that allows us to do a sort of gross

separation on the basis of size.

Gross meaning rough, not gross meaning bad.

Having that separation technique is very useful.

So for example, when we're working with proteins,

or molecules, metabolites, or any of these things,

they're coming from out of cells.

And if we want to isolate them from the cells

we have to do something about

separating the various components of the cells,

because separation is a means for isolating,

or what I call purifying, a molecule.

So if we want to purify a molecule

we really need to get that molecule

separate from everything else.

So what we will see in this process

is that there are numerous steps that have to happen

in order for us to be able to do that.

You'll see there are some shortcuts that work fairly well,

other times those shortcuts don't work well,

so there is no one way of isolating molecules.

There's no one way of isolating molecules.

Some molecules take a lot of steps,

some molecules don't take so many steps

and I'm hoping to equate you with some of the steps

in this process.

As I said, centrifugation is a way that we start

because we first of all have to get the material

that is part of the cell.

So the material that we're working with

might be in the cytoplasm of the cell,

the material we work with might be in the nucleus,

it might be in the mitochondrion.

Of course if we are talking about eukaryotic cells.

If we're talking about bacterial cells,

then it's basically in the cytoplasm

or it's in the membranes.

Of course it might be in the membranes

of eukaryotic cells as well,

so we have to have ways of isolating these various components

and centrifugation turns out to be a really, really good way

because these are very large complexes

and the way that we separate them is by what's called

differential centrifugation.

So differential meaning we use different rates of spinning

the centrifuge and those different rates of spinning

will cause different things

to go to the bottom of the tube.

So you can see here a depiction of some of those steps,

we've taken some a bunch of these cells

and the first step of isolating anything

is busting open the cells.

There are numerous ways of busting open the cells.

We can sonicate them, we can in some cases we can use enzymes.

In some cases we might want to use high pressure.

This particular method hears involves mechanical agitation.

It doesn't really matter.

First thing we do is bust open the cells.

We bust open the cells we've got the contents of everything

that's in that cell in that tube we're starting with.

You can see that here.

If we spin that at a low rate,

we will precipitate certain big things.

If we spin at a higher rate,

we'll participate smaller things.

So usually we'll spin it a slow rate at first

to get rid of the cellular debris,

the nuclei things like that,

and then we'll pour off the supernatant,

and take that supernatant, which is the liquid part

and spin it at a higher rate.

So when we do that we will spin out things

like mitochondria, etcetera.

Depending on where our protein or molecules of interest

are in the cell we can isolate

those components relatively efficiently.

Let's assume we have done that,

and now we've got this soup of whatever it is

that contains the molecules we're interested in working with.

One of the very common techniques that we work with,

and particularly it works very well with proteins for example,

and one that you probably did in basic biology or in chemistry

when you did things, was to use a technique called dialysis.

Dialysis is a way of separating small things

from big things and the basis by which it works

is it involves taking your soup,

your soup is the thing that you've taken out of the centrifuge

that contains the material that you're interested in,

and putting it into what's called a dialysis bag.

So we've done that here.

The yellow you see on the screen is the soup that we have.

The dialysis bag has a very useful property

that it is porous to the small molecules

that are contained in the soup

but it's not permeable to the larger ones.

So, for example I might have a soup here that is full of salt,

Salt sodium chloride, potassium chloride,

or something like that small molecules

and I want to get that protein and other things I have in here

separate from those small molecules.

The dialysis is very simple,

the small molecules as I said are permeable,

they'll come out, and the larger molecules are not permeable

and they will remain on the inside.

And in doing this I'm thus able to remove most of the salt

away from the proteins or the other things

that I'm interested in here.

So dialysis turns out to be a very simple

yet useful way of separating big from small.

There are other ways of separating big from small

That don't involve dialysis tubing

and they may involve separation of bigger molecules,

for example, that wouldn't fit through a dialysis tubing.

This technique, called gel filtration,

which is also called molecular exclusion.

Gel filtration equals molecular exclusion.

Involves a very cool trick.

Well as you'll see in these various techniques

that I'm going to show you,

the materials that are doing the separation

are typically used in what are called columns.

The material that does the separation

is actually shown over here on the right,

it's sort of an enlargement of what's in there,

and I'll tell you a little bit more about that.

So let's imagine-first of all let's talk about

what the material is, alright?

So let's say I've got a mixture of proteins

and I've got some proteins in there

that have a molecular weight of about 5,000

which wouldn't fit through a dialysis tube

and I've got other proteins that have a molecular weight of let's say 80,000

which also wouldn't fit through a dialysis tube.

So I can't use dialysis to separate those.

I've got to find some other way

to separate those proteins from each other.

Molecular exclusion or gel filtration

and by the way the word chromatography comes up,

when I say gel filtration chromatography,

all chromatography means is separation.

So gel filtration separation, molecular exclusion separation.

That's what those refer to.

These columns that I have here contain the material

that allows the separation to occur.

So as I said I want to tell you

a little bit about what these materials are.

As you can see on the enlargement

these are little round things.

This is a pretty big enlargement.

It turns out that these materials,

as we use them in the laboratory, are little tiny beads.

Little tiny beads maybe a millimeter or less in diameter.

They're pretty small.

But they have a really cool property.

The cool property that they have

is that they have holes in them,

and the holes are connected to tunnels within them.

And the tunnels go in one place and come out the other place.

So things can travel through these beads.

This technique relies on something very important

and the important thing that it relies on

is the fact that holes and the tunnels all have a fixed size.

They all have a fixed size.

That fixed size is called the exclusion limit.

Exclusion limit.

Why do I tell you that?

And why is that important?

Well, if they all have the same size,

the exclusion is that they're only going to allow

molecules of a certain size to fit into them

and travel through them.

So I gave you an example of 'I've got some proteins

'that have a size of 5,000 in molecular weight

'and some that have a size of 80,000 in molecular weight.'

If I used a set of beads that had an exclusion limit

of let's say 30,000 molecular weight,

what it would mean is that anything that has

a molecular weight of less that 30,000 will fit in there,

and anything that has a molecular weight

of greater than 30,000 will not fit in there.

Everybody got that?

So the exclusion limit determines

which things will fit in there.

Now, what does this mean,

well it turns out to be very simple.

Because if we think about the small guys going in here-

traveling through the column.

What we start with is a mixture of proteins,

we put them on the top of the column,

and then we take buffer and we let buffer flow

so that it's flowing through the column,

it's flowing through the beads,

and we're just simply monitoring

the rate with which the proteins pass through there.

And because one group of proteins is traveling through

the beads and the other group of proteins

is not traveling through the beads,

it means that the ones that are traveling through the beads

are in fact taking a longer distance through the column.

So therefore things that are less than the exclusion limit

will come out last.

Things that are greater than the exclusion limit

will come out first,

because they don't enter the beads at all.

Question?

The beads are in a fixed position, that's correct.

The beads are in a fixed position.

So buffer is flowing through them,

it's flowing through the column,

but for the most part we look at this thing right up here