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

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


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]