#06 Biochemistry Protein Purification Lecture for Kevin Ahern's BB 450/550


Uploaded by oharow on 07.10.2011

Transcript:
Captioning provided by Disability Access Services
at Oregon State University.
Kevin Ahern: So we're moving along nicely with the schedule.
And though we don't have to stay on it exactly,
we've been on it pretty good.
I've been pleased with the interactions
and also pleased with the questions I'm getting,
both in class and out of class.
So you guys seem to be engaging in this material
and that's a very good indicator of success.
So if you have questions, please feel free.
Come see me. Come see the TA's.
And we're here to help in any way that we can.
I only have one tiny little thing to say today
regarding the last of protein structure.
It's actually sort of an anecdote more than anything else.
And then I want to talk about techniques
for characterizing and/or purifying proteins.
One of the things that biochemists spend
a tremendous amount of time doing is just that: isolating,
characterizing, understanding proteins, enzymes, etc.
And so what you've learned so far about structure of proteins,
you will discover will be useful as tools
for learning how to isolate them.
And so I'll spend some time talking about that today
and also on Monday.
The anecdotal thing I wanted to mention to you
is the very last item on the protein structure page,
and it's actually this right here.
I've mentioned hydroxyproline to you already
and I want to reiterate something here.
Now, if you recall, I said that there are 20 amino acids
that we find commonly in proteins,
but we find modified amino acids in proteins.
And the point that I want to emphasize is that those
modified amino acids that we see happen post-translationally,
meaning that the modifications occur
after the amino acid is built into the protein.
So, in the case of hydroxyproline, for example, I gave you,
I showed you or described to you how Vitamin C
was involved in that reaction that modified the proline.
That happened after the proline
had been built into the protein.
The same is true of all the other things there are here.
Carboxyglutamate is an important modification,
as we will see,
that occurs as an important consideration in blood clotting.
Carbohydrate-asparagine adduct,
where we see, in this case,
addition of a carbohydrate to an asparagine residue,
this is really imporant in the production synthesis
of glycoproteins that we'll talk a little bit about later.
Phosphoserine, Phosphorylation is something
that you're going to hear a lot about later in the term
because phosphorylation is a means of controlling
or signaling through proteins.
And it's a very, very important mechanism for us to understand.
It's specifically phosphorylation
that I want to address briefly at the moment.
And that is that phosphorylation of amino acids
has to occur on side chains and side chains
that have hydroxyl groups.
So the three amino acid side chains that have hydroxyl groups,
of course, are the tyrosine, serine and threonine.
These are the three amino acids that get phosphorylated
or can be phosphorylated.
And we'll see a bit of a pattern
to how that phosphorylation occurs.
Not surprisingly, you might think,
well, why do these have such big effects?
You saw a big effect with hydroxyproline
because it was a part of that important structural
consideration for making a strong collagen.
In the case of phosphorylation,
what we're doing is we're converting,
excuse me, we're converting a side chain
from being hydrophilic to actually being ionic.
And so, in essence, what we've done is we've changed it from,
say, a partial charge to a fully negative charge.
In this case, we see two minus groups there.
Now, based on what I told you so far about protein structure,
you might imagine that changing the charge
of a specific location of a protein
might have structural considerations for that protein.
Imagine that previously we had a negative charge,
let's say a glutamic acid residue,
that was close to this proline
before we put the phosphate on there.
When we put the phosphate on there,
here's this negative charge before
that didn't really have that much interaction with the OH,
but now there's two minus charges over here.
What's going to happen?
Well, of course they're going to repel,
and when they repel,
that's going to change the configuration.
It's going to change the shape of that protein slightly.
And, as we will see,
and I've mentioned previously,
changes in the shape of proteins
can have some dramatic effects on the action of those proteins,
and we're going to talk more about those
as we get further along.
So those are some things that are other modifications
that can happen to proteins.
But I want you to be aware
that virtually any time you see a modified amino acid
in a protein it is because it has happened
after the amino acid has been put into the protein.
Okay, so that's the last of what I want to say
about general considerations of protein structure.
Now I'd like to turn our attention to characterizing proteins.
The first part of the characterization
I'll talk about is actually purification.
And purification isn't a spiritual purification,
but it's actually a physical purification.
I'll tell you a brief story.
When I was working in my very first lab after I had graduated,
I worked in a laboratory where we did HPLC,
and we had to have very pure solvents.
And I was very impressed by this notion
of purification that happens in there,
the need for purity in all biochemical materials.
And so I was very, very impressed
with these solvents that we used,
and we got them from this company that had purified stuff.
So I remember writing a letter to the companyótongue in cheek,
of courseósaying that, you know,
we found that not only were their solvents very pure,
but we had to do a spiritual purification
of these solvents before we used them, as well.
Of course, I wrote this as if it were completely serious,
sent it to the president of the company,
and, to my delight, I got this letter back
from the president of the company
congratulating me on describing for him
a new way of purifying his solvents that he could use for HPLC.
It was a good exchange.
So purification really had a big impact on me
as a very young biochemist.
Purification is important.
When we want to characterize,
let's say, a protein or an enzyme,
we need to have it isolated away from everything else.
When we try to understand an enzymatic reaction,
for example, we say, "Okay, well, ìI'm interested in this enzyme.
ìI'm interested in the reaction
that this enzyme catalyzes."
If I only have the soup of the cell, that is,
the cytoplasm of the cell that contains this,
I not only have that one enzyme that I'm interested in,
but I have several thousand other enzymes in there.
So it's important for me to understand what
this enzyme does that I be able to purify this
enzyme away from all those other proteins.
And so understanding how to purify one
protein apart from others is a very,
very important consideration in biochemistry.
Well, there are several techniques that
we use in order to do this,
and I'm going to go through and sort of describe
a few of the basic ones to you and then show you some
of the applications of these technologies.
You can't walk into a biochemistry
lab without finding a centrifuge.
It's almost impossible to do that and
that's because the use of centrifugal force as a means
of separating molecules on the basis of their size is a very,
very valuable tool.
Not surprisingly,
different things can be spun down.
We talk about "spinning them down."
That is, will they precipitate out of solution or
will they move to the bottom of the tube?
The function by which that,
by which they occur,
is a function of their size and the speed
with which we spin things.
So the largest things,
of course, as you might imagine,
spin most easily to the bottom.
So if I take and I'm interested in studying an enzyme in
E. coli cells, I can take a batch of E. coli cells
and I could use a fairly light
centrifugation and spin,
and those cells would come to the bottom of that tube.
Let's say I took that pellet which we get out of that,
and I'm interested in, not just the cells,
obviously, because I'm interested in
the enzymes that's inside,
I can use some techniques to bust 'em open.
I might use sonic waves to do that.
I might use enzymes to do that.
I might use mechanical agitation to do that.
It doesn't really matter the means I use.
But when I do that,
I basically open up the contents
of the cell and the insides spill out.
Those insides are going to have some things in them.
And, in addition,
I'm going to have some cell walls that are sitting there,
that now are empty of their contents.
I could spin those down again.
And if I did that,
I would basically have done my first separation.
I would have, on the one hand, the pellet,
which would contain the very big things,
like those cell walls,
and I would have the liquid component,
which would be the cytoplasmic material
that I was interested in.
I could take that cytoplasmic material.
I could do various centrifugations on it,
if I chose to.
And I could separate them on the basis
of the size of those complexes that are in there.
Now, we don't need to memorize numbers
or anything like that,
but I do want you to understand that centrifugation
allows us to do a sort of a rough separation based on size,
a very rough separation.
The stronger the centrifugal force,
the more things I'm going to pellet,
I'm going to drive to the bottom of the tube.
And there's a lot of different techniques
involved in centrifugation that allows me to purify things.
Now, centrifugation alone will notóunderline
"not"ógive me pure material.
So it's used mainly as a means of what
I would describe as fractionating.
When we fractionate things,
we break them into smaller pieces and
then we work with those pieces to do things of interest to us.
Let's imagine, for a moment, we've got two possibilities.
I took these E. coli cells
that I was describing to you,
and I'm interested in understanding a particular protein.
The first question I would ask is,
"Well, where's this protein?"
Is this protein in the cytoplasm?
Or is this protein embedded in the cell membrane?
Because both of those are possible.
The beauty of this is,
if I've fractionated it in this way,
I've got one fraction that has only things
in the cell membrane and I have another fraction
that has only things that's in the cytoplasm.
Then I can subdivide those further,
and that's some of the other things
I'm going to be describing to you.
So centrifugation, a very rough but powerful tool
to allow us to start to separate things
in the process of isolating components of cells.
Another techniqueóyes, question?
Student: Is size like actual physical size,
or is it like [unintelligible]?
Kevin Ahern: Yes, good question.
So is it actually the physical size?
Does density or mass play a role?
And all of these are variables in how things will separate.
So yes, those are factors,
especially as we get smaller and smaller,
some centrifugation techniques actually
work on individual proteins,
and what we discover with that is that proteins
that are very compact migrate through the centrifugal field
very differently than those that are very open.
So, yes, those are all considerations,
and we're not going to need to dissect those out,
but yes, you're correct, they do affect things.
A second technique that we would commonly use
in a biochem laboratory,
it's probably one you've played with in biology laboratories,
either in high school or college,
and that's dialysis.
Dialysis tubing is pretty cool stuff.
It is, basically, if you've never played with one,
it's basically a tube that is semi-porous.
It's semi-porous in the sense that it can allow water molecules
and small ions, for example,
to move through it, but larger things,
like proteins and DNA,
can't move through it.
And in biology labs we commonly use this as a way
of illustrating the concept of concentration and osmosis.
If I have a solution, for example,
that has a situation hereóhere's my cytoplasmic mix,
and let's say it's full of salt,
which I want to get rid of as much of the salt as I can,
I would put it into a piece of dialysis tubing.
The salt ions, the sodium and chloride,
are pretty small.
They will pass through the tube fairly readily.
The larger guys, my proteins and so forth,
won't pass through that tubing.
And after a period of time what I will see is that
the concentration of those salt ions inside
the tubing has decreased considerably as a result,
and, conversely, some water will actually enter that tubing.
And the reason it will enter that tubing is it's trying
to basically dilute out the things that won't come out,
that is, the proteins and so forth.
So I see a pressure that arises as a result of that.
Yes, Shannon.
Student: Isn't it, would it be impossible to actually get rid
of all the salt molecules?
Kevin Ahern: Is it impossible to get rid
of all the salt that way?
In theory, yes it is,
because I'm depending upon a differential concentration,
and even though I get it lower and lower and lower,
in theory, I could never get it completely out.
You're correct.
But this technique will give us a very nice simple way
of getting rid of a lot of small ions very readily.
And, in doing this,
I have actually increased the concentration
of my protein relative to the other things that are there.
A technique that is a useful technique that I'd like to describe
to you is that called "gel filtration"
and it's also called "molecular exclusion."
You see "molecular exclusion" over here.
"Gel filtration," we use the two terms interchangeably.
I actually sort of prefer "molecular exclusion"
but either one is acceptable,
as far as I'm concerned.
Now, to understand this technique,
we need to understand the sort of physical nature
of the separation.
So to use this technique,
I have to have something that's pretty cool.
So I have what's called a background
or matrix material which consists of millions
or I shouldn't say "millions,"
but thousands and thousands of tiny beads.
Little beads, maybe a millimeter or so in size,
big enough for your eye to see individual beads,
but they're still pretty tiny.
These beads have a characteristic.
The beads have little tunnels through them,
little tunnels.
And the little tunnels have openings
that are pretty uniform in size.
That turns out to be important.
So I've got a bead, I've got tunnels,
and the opening to those tunnels is uniform in size.
So to use this technique, what I do is,
I take my beads and I suspend them in a buffer.
So I suspend them in a buffer,
and the reason I want to use a buffer
is I don't want the pH to be too high or too low.
I want the protein to be stable,
because if I change the pH too much,
again, I'm going to denature it,
unfold it, and cause some problems.
So I have it in a buffer.
I take that sort of buffer containing these beads
and I sort of shake it all up and get it into a nice slurry.
Then I carefully pour it into a column.
And the beauty of this is that the beads,
of course, can't come through the bottom.
They get stuck right here.
And they form a column of beads.
So I've got thousands and thousands of these beads,
each with little tunnels through them,
each with a hole that's a set size.
And, yes, I can get beads with different
holes of different sizes.
But for any given experiment,
I'm doing one size of hole for one bead
and I've got thousands and thousands of those beads.
Once I have such a column,
I might run my buffer through it for a little bit,
just to make sure that it's washed
all the other junk out and so forth.
And then I've got a mixture of proteins that
I'm interested in separating on the basis of size.
This is a technique that allows us,
again, to separate on the basis of size.
The exclusion part of the technique goes as follows.
I've got in this mixture of proteins some that are
very, very large, maybe 200,000 in molecular weight or greater.
I've got some that are, let's say, medium size,
maybe 50,000 weight or greater.
And I've got some that are fairly small,
maybe 5,000 molecular weight.
And just as an example,
I just picked those three ranges,
What's going to happen with these three sets
of proteins relative to these beads?
Well, it turns out that the holes that I've chosen
in these little beads that I've got are such that
they will only let in things of a certain size.
There's a size exclusion.
So the great big 200,000 molecular weight proteins
won't fit in the holes.
They will not enter the beads at all.
The 50,000 are borderline,
they might be able to enter a few,
but they don't really enter very effectively.
And the 5,000 molecular weight proteins that I have
will basically see a hole and they'll go into it,
just because they can.
Well, if I apply these three to the top of the column
and then I let buffer sort of push everything through,
what I see is as follows.
The 200,000 molecular weight proteins will not enter
the beads and they will travel a very short path
through the column.
They just go shooting right through.
They're the very first thing that comes through the column
because they don't get distracted by going through all these
little tunnels on the way.
The 50,000 molecular weight proteins,
that can make it into some of those tunnels,
travel a slightly longer distance than the 200,000's do,
and consequently follow.
These would be the green ones on this display right here.
Last, the 5,000 will take the longest path
because they can virtually go through every tunnel
that they bump into.
So they take a much longer path going through the column.
So this column allows me to separate them on the basis of size:
the 200,000 guys coming out first,
the 50,000 molecular weight guys coming out second,
and the 5,000 molecular weight guys coming out third.
Now, as you can imagine,
when I have a mixture in cells,
I have all kinds of molecular weights,
so I don't just have three there, for example.
But you get an idea about the way that we can separate
on the basis of size.
So molecular exclusion is a very nice way of separating
these individual proteins and saying,
"Alright, I know my protein is around 50,000
"in molecular weight.
I can collect this fraction from around 50,000
and then work with it further to purify it."
Yes?
Student: How do you know when to...
the proteins obviously aren't actually yellow,
green and pink?
Student: How do you know when to switch,
that you're up to the next size?
Kevin Ahern: How do you know where they are?
They're not necessarily green or red or yellow.
It turns out that there's a couple of things that you can do.
One is, you can actually put molecular size markers
in there that are green or yellow,
which will help you.
But more importantlyóand your question's
a very good oneómore importantly,
I need to have a way of determining where my protein is.
That means I need to know something about what my protein does.
So I know my protein,
for example, catalyzes a specific reaction.
I could test each one of these and see
where is that reaction being catalyzed.
And so I say, "Oh!"
It appears over here in this tube,
so now I know that this is the range
where I want to collect my sample." Does that make sense?î
Kevin Ahern: And being able to assay what my protein does
is essential to purifying a protein.
If I don't, if I can't measure what my protein does,
I have no way of purifying it.
Yes, sir?
Student: Won't some of the smallest come out
with the biggest because
they don't all just go into the tunnels?
Some of it will just fall through normally, won't it?
Kevin Ahern: His question is, "How pure is this method?
Will you get a little bit of the smallest
with the largest?" Again,
it's kind of like the question Shannon asked about
being able to get rid of all of the ions.
Yeah, you will have microscopic amounts of things there.
This is not absolute purity that we're getting.
But, in general, you will see the smallest
will come out way, way late.
Yes, back there?
Student: How long does the process take?
Kevin Ahern: How long does the process take?
That's a good question.
It depends a little bit on the column.
Sometimes people really want to get
as much purification as they can,
and I've actually known people to pour columns
that are six feet high.
And those could take a few hours to run.
If I'm running a shorter one,
that might take an hour or two.
So it really depends upon what I'm trying to do
in terms of my separation.
But there are columns that people can pour that
are actually quite large.
Yes, sir?
Student: When you're saying that it's based on the size,
are you talking about physical size or the weight?
Kevin Ahern: Physical size and weight are related.
So, in general, when we talk about globular proteins,
even though they have individual shapes and so forth,
they, for the most part, have a given size per weight.
It's not absolute, but their growth,
as they get bigger in molecular weight,
their physical size will actually increase, as well.
So it's based on their physical size,
but since that's related to the molecular weight,
there's sort of a one-to-one relationship.
But it's not absolute.
Yes?
Student: Is this used to just primarily [inaudible] process?
Or is this ever done sequentially where you would take
a narrower range each time to evaluate a broad spectrum
of sample contents?
Kevin Ahern: I'm not sure I understand the question.
Student: Like, for each one of those,
if you took the yellow one that resulted from that,
and then put it back through another column
that had a narrower...
Kevin Ahern: That's actually a good question, also.
So could I take this guy and run it through
a different column that has a different size bead
that might be a little bit more selective in the process?
And the answer is, I could do that,
but there are other techniques that may be more useful to me.
And I'm going to show you some of those other ones.
But you're right, you could do that,
and take it over and say now you've got a smaller bead
and so you might be getting rid of some of the other
molecular weights that you don't want.
But, yes, you could.
One of the things that you discoveró
just a second, Shannonóone of the things that you discover
in purifying proteins is there's no one way to purify a protein.
Alright?
You have to adapt the methods
that you use to the protein itself.
And you don't know before you get started
what it's going to take to get that protein purified.
So there may be several different techniques
you'll have to use to get it,
and it's going to vary from one protein to the next.
Shannon?
Student: I was going to ask,
how do you know how often to change the tubes out?
Kevin Ahern: How do you know how often to change the tubes out?
Well, typically what people do with these
is they just count drops.
So I might say, "Okay, I'm going to get 50 drops."
If the drops are coming out at a reasonably even rate,
which they typically do,
then people will set up fraction collectors
so that every minute it will change a tube,
and that will have, on average, the same number of drops.
Paying somebody just to countóbelieve me,
I've done this myselfópaying somebody to count drops
before they switch the tube is one of the most
mind-numbing things that you can possibly have.
[laughter]
So this is one of the joys of automation in biochemistry,
when you've got a machine that will
automatically do that for you.
So that's molecular exclusion, gel filtration.
Another related techniqueóit's related only in the sense
that it uses beadsóis called "ion exchange chromatography."
So in this method, we also use beads,
as we used in gel exclusion, in gel filtration.
However, the beads don't have tunnels or holes in them.
Instead, the beads have on their surface chemical forms
that have been bonded to them
that have specific charge properties.
So what you see in this case is a set of beads that have,
on their surface, ionized, molecules that
when they ionize give negative charge.
When they ionize, they give a negative charge.
Now, these started outóhow do I take one of these?
I take my beads and the beads start out with a counterion.
I can't get a bead that has a negative charge on it
until I get it into solution and the ion comes off,
so typically the counterion might be,
in this case, a sodium.
I've got sodium ions out here
and they're attracted to those negative charges.
So I've got sodium ions mixed with these beads
and I've got them sitting in a bottle.
I take my solution, I take my buffer,
and I mix it just as I did before.
I pour my column just as I did before.
And those sodiums are still sitting there next
to those negatively charged beads.
Now I've got my proteins.
I've got my mixture of proteins.
Some of my proteins will have an overall negative charge.
Some of them will have an overall positive charge.
Some of them will have an overall charge
that's pretty close to zero.
So it's going to vary with the protein.
How many glutamic acids does it have in it?
How many lysines does it have in it?
And these are going to determine positive and negative charges.
Well, if I have beads that are mostly negative,
what will happen is, the proteins that
are the most positive will
actually kick off those sodium ions and replace them.
This is the "exchange" part in the name.
They're exchanging those counterions,
in this case, the sodium ions.
So the positively charged proteins will kick off
the sodium ions and the positively charged proteins
will "stick," quote-unquote, to that bead.
What's going to happen to the negatively charged proteins?
Well, guess what?
They're going to come shooting right through,
because they don't want to interact
with these beads, at all.
So what I've done with this technique
is I've separated proteins on the basis of their charge.
The most negative ones are going to come racing off.
Those zero ones are probably going to follow that.
And then the positives are going to follow that.
And you might say,
"Well, why do the positives even come off at all?
Or how do I get the positives off?"
That's one of the most common things.
If I want the positive ones,
you know, I've got them stuck to the beads.
How do I get them off?
The answer is this.
Virtually every kind of interaction we talk about in this class
is not a covalent interaction.
These are attractive things.
So if I can make something else replace those proteins,
I can get the proteins to come off.
It turns out, if I pour a concentrated sodium
chloride solution in there,
there's enough sodium there it will displace
those positively charged proteins
and then I can get the positively charged proteins off.
So there's an exchange.
First, the protein displaces the sodium.
Then high concentrations of the sodium
will displace the protein, and I've got what I want.
So I've separated my proteins on the basis of charge.
This particularly phenomenon I've just described to you,
in general terms, is called "ion exchange chromatography,"
but more pecifically, this is called
ìcation exchange.î
Cations, of course, refer to the positively charged ions,
and what's being exchanged were those first sodiums.
They were positively charged.
This is cation exchange chromatography.
So in cation exchange chromatography the first guys
that come off will be the negatively charged proteins.
The last ones to come off
will be the positively charged proteins.
Is there an anion exchange chromatography?
You betcha.
So if I have anion exchange chromatography,
instead of having beads that are negatively charged,
I have beads that are positively charged.
And exactly the opposite of everything
I've just said is the case.
Instead of having sodium as a counterion,
they'll have chloride as a counterion.
And the chlorides get displaced
by the negatively charged proteins.
The positives, of course, come racing through.
So we just flip everything backwards if we have anion
versus cation exchange chromatography.
Yes, sir?
Student: Regardless of whether you're using anion
or cation exchange chromatography,
wouldn't your initial sample received
also include the neutral?
Kevin Ahern: So the sample will also include the neutral
and it will come out somewhere in between the two.
Yes, it will.
Remember, we've get a whole, we've got thousands
of proteins in here.
We've got a lot of different proteins.
So we're going to have sort of a spectrum,
some with a lot of negative charge,
some with a little bit of negative charge,
some that are zero,
a little bit of positive, etc.
And that actually is going to relate to another technique
I'm going to talk about in a minute.
But you're right.
There's a whole spectrum of these that are there.
So, again, we're talking about techniques that give us basic,
simple ways of separating things.
But they're not absolute.
I don't get only the one thing I want there.
I've got some other components that are there.
And there's no technique that I will tell you
that is going to give you absolutely one thing.
Understand that. That's important.
If you wonder what those anion versus cation
exchangeóyou don't need to know these structures,
I'm just showing it to youóhere's an example of something
that would have negative outside.
It's got a carboxyl group on there.
Here's something that might have
a positive thing on the outside.
You can see this tert-,
uh, quartern-, amine that's out there,
actually a tertiary amine that's out there.
And these are commonly used,
but, again, don't worry about the structures of those.
One of the more powerful techniques that's used in a laboratory
for purifying proteins is called "affinity chromatography."
So, like the other two techniques I just described,
it also uses beads.
But instead of having tunnels or
instead of having charged molecules,
this technique uses specific chemicals on the exterior.
So to describe this I need to give you an idea
about how I might use this technique first.
Let's say I'm studying,
I'm going back to my E. coli cells
and I'm very interested in a protein
that I know binds to ATP.
I know it binds to ATP because it uses it in a reaction
that it does.
So I know that this protein will bind to ATP,
What I do is I take this naked bead
that doesn't have anything else on it,
and I treat it so that chemically it is bound to,
covalently stuck to, ATP.
So I can covalently link ATP to a naked bead,
as it were.
So now I've got all my beads and they each have hundreds
or thousands of ATPs stuck,
just out here,
facing the solution, in the bead.
Well, now I take this mixture of beads
that all have ATPs on them,
and I pour my column with my buffer,
as I did before.
And now what's going to happen is proteins
that bind to ATP are going to stick to this column,
and proteins that don't bind to ATP aren't going to stick.
Well, this is a really powerful technique,
a very, very powerful technique.
Will I only get proteins that bind to ATP?
Well, I might get a little bit of other stuff,
but for the most part I'm going to get proteins
that bind to ATP.
Is that only going to be one protein?
Well, no. There are many proteins in a cell that will bind to ATP,
but I'll have a nice collection of the ones that do,
and my protein's going to be one of them.
Yes, sir?
Student: Can a bead get more than one ATP on it?
Kevin Ahern: Yes. Can a bead get more than one ATP on it?
It can get thousands,
Yes. Yeah.
Well, how do I get myójust a second,
Shannonóhow do I get my protein off?
I would ask you that question.
How would I get my protein off of such a column?
What would I have to add?
Student: Whatever the natural [unintelligible] is.
Kevin Ahern: ATP.
I could add ATP, right?
And so now my protein's going to let go of this
and it's going to grab ATP and it's going to come off, right?
That's a very cool thing.
Because, again, remember, the protein is not covalently bound,
so it's going on, going off, going on, going off.
And when it comes off,
a loose ATP comes in here,
it binds to ATP and now it comes off the column
and doesn't stay stuck.
So I add the natural ligandóin this case,
ATPóto the molecule.
Shannon, did you have a question?
Student: Yeah.
Is it practical to functionalize your beads?
Or do you usually buy them pre-functionalized?
Kevin Ahern: Yeah.
Is it practical to functionalize your own beads,
or do you buy them pre-functionalized?
You can do both. So it depends.
If have something that's a very specific molecule,
you might do it yourself.
Good questions.
Alright, so affinity chromatography is really a very nice way
of doing purification for specific target proteins.
I want to just briefly mention one other
because you frequently see it in laboratories.
It's called HPLC, and HPLC stands foróand this is commonly
misstatedóhigh performance liquid chromatography...
high performance liquid chromatography.
A lot of people say high pressure liquid chromatography
because the columns generate a lot of pressure,
but, in fact,the correct name is high performance
liquid chromatography.
This is a technique for separating,
usually, fairly small molecules.
But even that's not absolute.
That's been adapted somewhat over the years.
The way that this technique works is by taking and,
instead of using a nice glass tube that's there,
these are typically poured into stainless steel tubes
that have great strength.
And the reason they need great strength
is because these are used to,
at very high pressure.
You don't want them to burst, for example.
Well, what's the packing material?
The packing material here is also beads,
but the beads are microscopic.
They're very, very, very tiny.
So they're smaller, an individual bead would be smaller
than your eye would recognize.
They come as powders, essentially.
And these powders have on them long hydrophobic sections
of molecules, like long fatty acids, for example.
A commonly used one is called a C-18.
And what that means is that the bead
has a whole bunch of 18-carbon units with hydrogens on them,
sticking off...
very, very hydrophobic.
So now what I have, because the beads are so tiny,
is I have millions of interfaces,
millions of these hydrophobic molecules that the solvent
is in contact with.
If I pass my material through it,
first of all, to get it through,
it takes high pressure because these things are packed
very, very densely And they're packed densely
so I can get as many of these
possible things in there as I can.
Well, now, instead of having charges,
or holes, or specific affinity molecules,
now I basically have a bed,
alright, that is the column material,
I have a bed of hydrophobic side chains.
What do you suppose is going to stick to it?
Well, the things that are going to interact with those
hydrophobic side chains are going to be hydrophobic molecules.
And the things that are not going to interact
with that support are going to be hydrophilic.
So now I can separate on the basis
of whether something likes water or doesn't like water.
The ones that will come off of a column like this first
are the hydrophilics because they don't interact
with those C-18 groups.
The ones that are going to come off last will be those
that are hydrophobic,
that do interact with those.
The rate with which they come off is actually
a function of their hydrophobicity.
So, again, we can imagine a range of things,
that are very hydrophilic,
very hydrophobic, and things somewhere in between.
What I've just described to you, and, by the way,
there are a couple different strategies for HPLC,
but what I've just described to you is the most common form,
and it's the only one you're responsible for.
It's called "reverse phase chromatography," reverse phase.
Now I want to spend a few minutes telling you
about a couple of techniques that now get into some
really cool stuff with respect to purification of proteins.
I'm going to skip down,
and I'll come back and talk about
polyacrylamide gel electrophoresis later and SDS.
What I want to talk about right now
is an interesting technique called
"isoelectric focusing."
Isoelectric focusing is a little difficult to conceptualize,
but I'll try to do it here.
Imagine, if you will, I now have a bunch of beads.
And these beads have,
not one property,
but they're a mixture of beads,
each with their own property.
So before, I used all the beads that had the same hole,
or they all had the same negative charge,
or they all had the same affinity molecule,
or they all had the same C-18 group.
Now, I have mixtures of beads,
each with their own property.
What's the property?
Well, the property is as follows.
Some beads will have on them,
let's say, 50 negative charges.
And some beads will have on them,
let's say,
49 negative charges.
And some will have 48,
47, 46, 45.
I go all the way down to zero.
And then I have some beads that have +1 charge,
and some that have +2,
and some that have all the way up to +50,
just as an example.
Everybody envision that?
So I've got some beads that have all these different things.
So I take this slurry of all these beads and I shake 'em up,
and I put them into tube,
a glass tube, as I did before.
And these beads are relatively mobile.
That is, they can move around.
They're not like the column I did before.
Instead of standing it up like this,
I lay it out like this.
And now I apply an electrical current to it.
What's going to happen?
Well, to the positive end,
the most negative charged ones are going to race
and get over there, right?
And at the negative end,
the ones that are the most positively charged
are going to race and get over there.
And right square in the middle,
those that are zero are going to stop right there.
That make sense?
So what I've just made in this tube is a gradient of charge...
a gradient of charge,
from the most positive at one end,
to the most negative at the other end,
with zero in the middle.
Everybody envision that?
So this is called "isoelectric focusing."
It turns out that what I have just described to you,
in terms of separating charge,
also separates on the basis of pI.
We talked about pI.
pI is the pH at which a molecule has a net charge of zero.
And so by setting up a column like this,
I actually separate molecules on the basis of their pI,
the pH at which they have a net charge of zero.
The ones that have the lowest pI's will be at one end,
the ones that have the highest pI's will be at the other end,
and the ones closest to a pI of 7 will be right in the middle.
Everyone with me?
Well, to do this kind of experiment,
to do this kind of a separation,
I take not just the beads,
but I take all my proteins and I mix it with the beads.
I take all my proteins and I mix it with the beads.
My proteins have a variety of charges on them.
Some are very negative,
some are very positive,
and some are somewhere in between.
When I apply the current,
just as the beads separate themselves,
so, too, do the proteins separate themselves...
one end very low pI,
one end very high pI,
in the middle,
those that have a pI around 7.
So I've separated all of my proteins on the basis of their pI.
Yes, sir?
Student: Do you really need the beads?
Kevin Ahern: Yeah.
It's a good question.
I do need the beads because the beads provide a support.
In theory, I wouldn't need to do that.
But if I don't have the beads there,
the proteins just come racing off.
So, yes, I do need the beads there.
Yes?
Student: In this slide,
does [unintelligible] stand for pI?
Kevin Ahern: No. It's a pH gradient.
And because it's a pH gradient,
that's where the pI's line up.
So at a given pHóthat's a good questionóbut at a given pH,
if the pI of this molecule is, let's say,
3.2, that means that molecule has a net charge of 0
right here and that's why it migrates to that point and stops.
Does that make sense?
Student: So, like, everything with a low pI
would be towards the positive end
and everything with a high pI would be towards the negative end?
Kevin Ahern: Actually, it's backwards of that.
But, yes.
But you don't need to worry about that.
All I want you to know,
at this point,
is that it is simply a separation on the basis of pI.
Yes, sir?
Student: So are the beads small,
like the powder?
Are you trying to pack as many in there as you can?
Kevin Ahern: Are the beads that small?
No, the beads are not very small.
The beads are relatively large.
Student: On the top picture,
I don't understand,
like, that there's the plus,
there's the plus/minus and the minus
unintelligible] the three colors.
Kevin Ahern: Well, this is just simply saying that,
here these guys are the most positive.
They're going this direction.
These are the most negative.
They're going this direction.
And the in-betweens are going to be in here.
That's all that's saying.
So, keep it simple.
Keep it simple.
So we've got positive,
negative and basically neutral in the middle.
I've got a gradient of that,
So this is a way of separating proteins on the basis of pI.
Now this, in itself, is useful.
For example, I say, "Well, my protein has a pI of about 3.2,
I could go and cut out the band that corresponding to 3.2,
and I would have a mixture of proteins
that all have similar pI to my protein,
right?
That's not the most important or the most
powerful application of this technique.
But in order for me to understand a more,
for you to understand a more powerful application,
we have to understand this process first.
So I'm separating on the basis of their pI's.
I have a whole gradient of pI's.
What's the next thing I do?
Well, next time I'll tell you a little bit about gel separation,
but I'm going to cheat and tell you about gel separation here,
Now, keep in mind what I just told you about
isoelectric focusing.
We're going to use it in a second.
But before we get to apply this technology into something else,
we need to understand how we separate proteins.
How many people here have ever run a gel in a laboratory?
Many people have.
Gels are ways of separating molecules using electricity
on the basis of their size.
I'll talk about the theory for that in the next lecture,
but today all we need to understand
is that gel electrophoresis,
as it's called,
separates molecules on the basis of their size.
The largest ones are the slowest moving
and the smallest ones are the fastest moving.
It uses electricity to do it.
As you might imagine,
it involves charge.
We'll talk about the specifics next time,
but we're going to have gel electrophoresis separating proteins.
So if I take my mixture of proteins
and they've got a whole bunch of sizes and I apply them
to the top of the gel,
what will happen is,
the electricity will drive them through,
with the smallest ones moving the fastest and the slowest ones,
or the biggest ones moving the slowest.
Now, here's the clincher,
and this is the cool thing.
The cool thing is,
I can combine these two technologies.
I do something called two-dimensional gel electrophoresis.
It's schematically shown here.
The two dimensions are,
I do two different techniques.
First, I take my mixture of proteins and I mix it with this
slurry to do isoelectric focusing.
So I take my tube.
I lay it out here.
I apply the current.
I get the separation on the basis of pI.
So I have this tube now that has this gradient
of proteins separate on the basis of their pI.
Alright?
I'm very careful and I slice open this tube,
and I take that material that's in there
and I put it on the top of the gel.
And now I run electric current through the column material
and driving those proteins into the gel,
first I separate it in this dimension on the basis of pI.
Now I'm going to separate all those guys on the basis of size.
What I will see is something that
schematically looks like this.
So, if I were to look at this,
the molecules that have the most positive charge
will be on the left side of this gel.
The ones that have the most negative charge
will be on the right side of this gel.
And those that are the largest will be on the top,
and those that are the smallest will be on the bottom.
Down here, I would expect proteins would be small,
positively charged.
Over here, I would expect proteins would be large
and negatively charged.
Now, in two dimensions,
I can separate every protein in this cell.
Every protein that was in my mix I can now separate
and actually see a spot on this gel.
Let me show you what this looks like.
This, I think, is a magical technology,
This is what one might look like.
Now, we see quite a bunch of interesting stuff here.
We see dark bands.
We see light bands.
We see all kinds of mixtures of stuff.
But, again,
largest and most negative...
smallest and most positive.
Neutral, small.
Neutral, large.
Really interesting stuff.
You say, "Well, that's cool.
That's really totally there for a nerd." Right?
Only a nerd could love the beauty in one of these things.
And I'm going to make you love 'em,
too, Which basically means I'll make a nerd out of you,
alright?
The beauty of thisólet me finishóthe beauty of this is that what,
let's imagine, if you would,
that I'm a person who is a medical doctor.
And I've got a patient who has a liver tumor,
And I want to understand how the liver tumor proteins
are different from the proteins in the non-tumorous
part of the liver.
I could operate.
I could remove that tumor.
And as I'm removing that tumor,
I could scrape off some normal cells
from that same person's liver and I could isolate
the proteins from each.
And then,
I could do a 2D gel on the normal liver cell proteins
and I could do a 2D gel on the tumor cell proteins,
and, guess what?
I'm going to see differences.
These are reproducible.
So I could look and say,
"This band right here,
look how intense that is in the tumor cell.
I don't hardly see this protein,
at all, in the normal cell.
Here is a protein I see in the normal cell.
I don't see it in the tumor cell." I could understand,
for every protein that's in these cells,
I could understand whether it's more in tumor,
more in normal,
or no difference.
I could understand,
at the protein level,
one of the mechanisms and one of the differences
between a normal cell and a tumor cell.
And I could do it in a single gel.
That's absolutely phenomenal!
Let's imagine that you're a pharmacist.
I'm not quite done, yet.
I'll be done in just a second.
Let's imagine that you're a pharmacist and
you want to test a new drug that your company has just created.
What's the effect of this drug?
Are there any nasty side effects of this drug?
Well, I take one group of cells.
I treat 'em with my drug.
I take the other group of cells.
I don't treat them.
And I compare.
"Oh, my god!
This thing's knocking down DNA polymerase tenfold!
I'd better be careful with this stuff." Alright?
"This thing isn't having any effect,
whatsoever." Maybe I'm interested in a compound
that somebody says, "Hey!
It's carcinogenic."
It really affects cells if I have this.
One treated, one untreated,
and I can look at the entire pattern of proteins...
an absolutely phenomenal technology.
Alright.
That's enough for today.
I'll see you guys on Monday.
Student: So do they have this stuff archived?
Kevin Ahern: Do they have these?
There are many places where you can archive this information.
Student: So you can, like, do matching that way?
Kevin Ahern: You can, but, in general, you'll want to do it yourself,
just to make sure that there's not variability from that.
Student: It must be really hard.
Kevin Ahern: It's a sophisticated technique, yeah.
Yes, sir.
Sorry.
I wanted to get through there.
Yeah.
Student: That's That very top left corner, marked negative?
Kevin Ahern: Uh-huh.
Student: Well, was that a natural protein sample,
would you think?
Or is that an artifact from the actual process?
Because it was a large smear.
Kevin Ahern: Smears will happen when you've got things that don't fit in well,
and they're actually artifacts,
in a sense, but they're real things,
but they're not [unintelligible].
Student: Do they have a "bible," if you will,
of different,
like...
Kevin Ahern: They do.
They do.
Isn't it cool?
Yeah.
Did you have a question?
Student: [unintelligible]
Kevin Ahern: Yes.
Student: Is there a standard that you can look at [inaudible].
Kevin Ahern: Very good question.
That's what everybody else has been asking.
Kevin Ahern: So, yes, there are,
and if you remind me,
I'll say that at the beginning of the lecture next time.
There are libraries of these where you can actually
do that comparison, which is kind of cool.
Excuse me.
Oh, I'm sorry.
Good day.
How are you doing?
I've gotta squeeze in here.
Sorry.
Student: Sorry.
[END]