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  • Another Monday beckons, another week beckons.

  • One day closer to an exam.

  • Student: Whoo!

  • Kevin Ahern: Yay, huh?

  • One day closer to your opportunity

  • to show me how much you know.

  • That's good.

  • I hope you had a good weekend.

  • Student: Fantastic.

  • Kevin Ahern: Fantastic?

  • Student: We won.

  • Kevin Ahern: Are we talking about football here?

  • Student: Yeah.

  • Kevin Ahern: Okay.

  • So the football team won.

  • So last time, I threw out the topic

  • to you of the 2D gel electrophoresis,

  • and I think that's a really phenomenal technology.

  • I think it allows

  • not "I think", I know it allows

  • us to do amazingly complex analyses of cells.

  • And if we have cells that have different experiences

  • one being a tumor cell, one not being a tumor cell,

  • one being treated with a drug,

  • one not being treated with a drug, one being starved,

  • the other not being starved, et cetera, et cetera

  • we we can use this technology to see

  • very clearly at the protein level

  • how these changes occur inside of the cells.

  • Several students after the class asked me if there were

  • libraries of gels that were out there that are cells

  • of known treatments.

  • The answer is, there are.

  • But many laboratories will actually do their own

  • side-by-side comparison because one of the things

  • that you see is the reproducibility is not 100% the same,

  • so if you've done both of them in your

  • laboratory at the same time,

  • you're a little bit more able to compare them.

  • So that's something that happens.

  • But, yes, there are libraries of such things out there.

  • And I just realized, I haven't checked the camera

  • to make sure it's properly on the screen.

  • So give me just a second to check that.

  • Doo-do-doo-doo.

  • And the answer is, it was perfect.

  • Alright.

  • There's nothing worse than looking at your video afterwards

  • and you see you had it about halfway on screen

  • and about halfway off the screen.

  • And you guys like that about as much as I do,

  • so, yeah, maybe less than I do.

  • One of the things I skipped over in getting to tell you

  • about 2D gel electrophoresis was to tell you about

  • gel electrophoresis itself.

  • So that's how I'm going to start the lecture today,

  • telling you how gel electrophoresis works

  • and I'm going to talk about

  • two different types of gel electrophoresis.

  • The first type I will talk about

  • is actually the simpler of the two,

  • and it is what we refer to as DNA,

  • separating DNA by agarose gel electrophoresis.

  • Agarose is, and there's the word right there

  • agarose gel electrophoresis,

  • I keep popping out here

  • agarose gel electrophoresis is a technique.

  • I don't have a figure for it anymore.

  • Your book used to have a figure and then they took that

  • away from me, so I don't have the figure out for it.

  • But I can tell you it's, in principle,

  • very much the same as polyacrylamide gel electrophoresis.

  • So let me just show you what that looks like.

  • Agarose gel electrophoresis is what we use

  • to separate fragments of DNA.

  • We can also separate fragments of RNA with it.

  • We do not use agarose gel electrophoresis to separate proteins,

  • and you'll see why that's the case in just a little bit.

  • The first reason, though, that we don't use it to separate

  • proteins is that nucleic acids are way bigger than proteins.

  • The biggest molecules in the cell are DNA molecules, by far.

  • Proteins don't even come close in terms of size.

  • What the agarose provides, in the case of DNA separations,

  • or what the polyacrylamide provides,

  • in the case of protein separations, are a matrix.

  • And we can think of this matrix

  • sort of like it's schematically shown here.

  • The matrix is a series of strands or connected

  • things that provide a support.

  • The support is to support the liquid of the buffer.

  • So just like we could take a mix of Jello

  • and put it into water and boil it,

  • when it cools down, it forms a solid support based

  • on what was in there, so, too, can we do with materials

  • for the gel, the difference being, in the case of a gel,

  • that these strands that provide the support will provide

  • little channels or little holes through

  • which the macromolecules can elute.

  • And I'll show you how that happens, okay?

  • Agarose has bigger holes than polyacrylamide does.

  • So we need those bigger holes to separate DNA molecules.

  • So how do I separate using gel electrophoresis for DNA?

  • Well, first of all, I take my DNA molecules

  • that would be a mixture of different sizes.

  • And I would apply them to the top of my gel,

  • as you can see here.

  • So I make these little indentations

  • that are what are called "wells."

  • And into these wells, we pour our mixture of DNA fragments.

  • DNA fragments are negatively charged.

  • They're polyanionic,

  • meaning that they have many, many negative charges.

  • For every base that we add, we get another negative charge.

  • So the charge is proportional to the length,

  • and the length is proportional to the length.

  • Now, you'll see why that sort of makes sense, in a second.

  • The charge is proportional to the length,

  • and the length is proportional to the length.

  • And what we do in separating these guys

  • is we use an electric field.

  • The electric field we use places a negative charge at the top.

  • You can see that little negative ion right there.

  • And it places a positive charge at the bottom.

  • The DNA molecules, being negatively charged,

  • are repelled by the negative at the top

  • and attracted toward the positive at the bottom.

  • Well since the ratio of the charge to size is constant,

  • that is the longer molecules have more charge,

  • but they also have more size

  • the separation that happens between these molecules

  • is solely on the basis of their size...

  • solely on the basis of their size.

  • The smallest guys can move the fastest through

  • these channels and they go racing through the gel.

  • The largest molecules don't have that same mobility

  • and it takes them longer to get through the gel.

  • So at the end of a stint of gel electrophoresis,

  • what we see is the gel products.

  • So this is a protein gel,

  • but a DNA gel would look very much like this,

  • where we have fragments that have been separated by size.

  • So this would be the largest molecules up here.

  • These would be the smallest molecules down here.

  • And these are specific fragments, in this case,

  • that have been purified of a protein

  • that have a given size that's there.

  • So, in principle, DNA electrophoresis and protein

  • electrophoresis are the same after we have to do some

  • manipulations to proteins to make that happen,

  • and I'll show you how that occurs.

  • So DNA electrophoresis makes sense?

  • Yes, sir?

  • Student: So if the charge on the bottom isn't

  • great enough that it's, it's not just going

  • to tear through the gel?

  • Kevin Ahern: So his question is the charge on the bottom

  • great enough that it's just going to not tear through the gel?

  • In fact the molecules will, if you leave it long enough,

  • go all the way through the gel.

  • Yes, they will.

  • So they will go all the way through,

  • this is cutting out.

  • They will go all the way through the gel.

  • So there's several variables that we have.

  • We don't need to consider them really here,

  • but I will tell you we can change the percentage of agarose,

  • which will actually change the size of those holes

  • that the DNA molecules are passing through.

  • So we can optimize that for different

  • things that we're trying to separate.

  • And I'm getting some noise.

  • Maybe that took care of it.

  • So that's DNA electrophoresis.

  • It's pretty straightforward.

  • With protein electrophoresis,

  • we've got a different consideration.

  • And the reason we've got a different consideration is,

  • first of all, proteins are globs.

  • And second of all,

  • proteins don't have a uniform mass-to-charge ratio.

  • Some proteins are going to be positively charged.

  • Some are going to be negatively charged.

  • Some are going to be neutral.

  • And that charge is really unrelated to the size of the protein.

  • So if we try to separate proteins without some other things

  • to give an artificial size-to-charge ratio that's constant,

  • then we're going to have trouble.

  • Because if I take my mixture of proteins

  • and I've got some positive ones on top

  • and some negative ones in there,

  • the positive ones aren't even going to enter the gel.

  • They're not even going to go in.

  • Boy, this is really misbehaving today.

  • Alright.

  • So I have to do something, then, to make the,

  • I have to do something to make the proteins have

  • a reasonably constant charge, or size-to-charge ratio.

  • So the trick that's used is a very clever one

  • and it works very, very well.

  • It may seem a little odd, at first, but it's actually a very,

  • very good way to give proteins an artificial

  • size-to-charge ratio that's constant.

  • What we do is take the mixture of proteins

  • that we want to separate,

  • and we add excess detergent, called SDS.

  • That stands for "sodium dodecyl sulfate."

  • So it's a long carbon chain molecule that has

  • at one end a sulfate.

  • Now, that sulfate is negatively charged.

  • When these proteins encounter the SDS,

  • if you recall when I talked about

  • what detergents can do to protein,

  • what did I say would happen?

  • They denature, they unfold.

  • So this protein that starts out as a glob,

  • first of all, elongates out into a nice long chain.

  • So, visually, we could imagine this guy is going to look

  • something like a straight DNA molecule,

  • not as big, but a straight DNA molecule.

  • The second thing that happens is these

  • sodium dodecyl sulfates completely envelope the chain.

  • Alright?

  • They just completely go all the way around the thing,

  • making like a Twinkie or something, okay?

  • A Twinkie's got the little

  • chewy center, right?

  • The chewy center being the protein,

  • and it's got this coat of stuff all the way around it.

  • Well, that coat, of course,

  • is proportional to the length of the polypeptide chain.

  • Longer polypeptide chains will have more of those

  • sodium dodecyl sulfates than smaller ones will.

  • So the size-to-charge ratio is relatively constant.

  • It's not absolutely constant,

  • but it's relatively constant.

  • And, in fact, for most purposes,

  • it's constant enough that we can get very,

  • very good separations based on size.

  • So once we've done that,

  • we take our mixture of proteins,

  • that are now all coated with this SDS,

  • and we separate them on a polyacrylamide gel.

  • And as I said earlier,

  • the only difference between agarose

  • and polyacrylamide is that polyacrylamide

  • simply makes smaller pores,

  • smaller holes, for those proteins to go through.

  • We apply an electrical current,

  • just as we did before,

  • negative at the top,

  • positive at the bottom,

  • and we separate solely on the basis of size,

  • how fast they can move through that chamber.

  • Now, and so when we do that,

  • we actually end up getting a gel.

  • This actually is a protein gel.

  • We can see these are marker proteins that have different sizes,

  • the largest ones being up here,

  • the smallest ones being down here.

  • And if we know the sizes of these known proteins over here,

  • we can actually determine the size of an unknown protein

  • by seeing where does it line up with.

  • Is it 50,000 in molecular weight?

  • My protein must be about 50,000 in molecular weight.

  • So this technique has an acronym.

  • First of all,

  • polyacrylamide gel electrophoresis has the acronym PAGE,

  • P-A-G-E.

  • When I use SDS,

  • which I almost always do with proteins,

  • we call it SDS-PAGE.

  • SDS-PAGE.

  • So SDS-PAGE allows me to separate proteins

  • on the basis of their size,

  • very much like I separate DNA molecules

  • on the basis of their size.

  • So when I'm doing that 2D gel electrophoresis that

  • I talked about on Friday, that second dimension,

  • or the first dimension,

  • we had isoelectric focusing,

  • and I said we cut it open,

  • and we laid it on top of this gel?

  • Well, this gel is a polyacrylamide gel.

  • We have to make sure we get some SDS in there

  • so the proteins all make the Twinkie shape,

  • right?

  • And then we run them through in that

  • second dimension as SDS-PAGE.

  • So the first dimension of a 2D gel is isoelectric focusing.

  • The second dimension is SDS-PAGE.

  • And thanks to that,

  • we can actually separate these molecules and determine,

  • literally, the amount and presence or absence of virtually

  • every protein that's made in a given cell.

  • Student: So that SDS part,

  • you just call that isoelectric focusing?

  • Kevin Ahern: What's that?

  • Student: Is that SDS part,

  • you just call that isoelectric focusing?

  • Kevin Ahern: No.

  • Isoelectric focusing is a different technique.

  • That's the one where we used the charge to separate

  • the molecules in the tube?

  • Student: For DNA.

  • Kevin Ahern: No.

  • That's for protein.

  • That's what I talked about last time.

  • Isoelectric focusing,

  • I put the stuff in the tube,

  • and I had the things that had minus 50 all the over to plus 50?

  • Right?

  • So that first dimension,

  • I separate on the basis of what was essentially the pI.

  • Okay?

  • And the second dimension I separate on the basis of size.

  • That second dimension is known as SDS-PAGE.

  • Yes, sir?

  • Student: So the SDS coat doesn't affect so much

  • with the isoelectric focusing?

  • Kevin Ahern: Okay.

  • That's a good question,

  • a common question.

  • So people will frequently say,

  • "Does it screw up the isoelectric focusing?"

  • Well, no, because they've already been separated

  • by the isoelectric focusing.

  • And so we're just covering what's already been separated

  • on the basis of pI with SDS.

  • It doesn't screw anything up, at all.

  • If it did, we would have a problem.

  • Student: Okay.

  • So they're not actually run at the same time?

  • Kevin Ahern: They're not run at the same time,

  • because we have to separate on the basis of pI first.

  • That would be a good exam question

  • if we tried to put the SDS in with the isoelectric focusing,

  • what would happen is, everything would be negatively charged.

  • It would all go to one end.

  • Good question.

  • Yes, Shannon?

  • Student: So how is it physically transferred to the PAGE?

  • Kevin Ahern: It's just laid on top of the gel.

  • It's just, so instead of having individual wells,

  • I would just have a long thing.

  • I'd lay my little tiny tube up there.

  • And I would just lay it on there

  • and run electrical current through it.

  • Yeah.

  • The people who run 2D gels, it's an art.

  • Believe me, it's an art.

  • So getting that little tube on there

  • and not breaking it and fracturing it and everything,

  • so that it lays evenly across the gel surface,

  • is very important, and there really is an art to it.

  • Yes, sir?

  • Student: I've seen agarose gels for DNA that are relatively small.

  • Is there a standardized size for these?

  • Or is there a variation in size?

  • Kevin Ahern: His question is,

  • do we have variations in polyacrylamide

  • that we use for protein gels,

  • because we do see variations that we use for agarose.

  • And the answer is, yes, we do.

  • So we can run polyacrylamide gels varying the percentage

  • of polyacrylamide that's there and also varying the number

  • of links between the individual strands.

  • And that is based on the chemistry that's involved

  • in making a polyacrylamide gel.

  • And all we're doing, in either of those,

  • is really determining the size of those holes

  • that the proteins are moving through.

  • So we can adjust that.

  • If we have a bunch of small proteins,

  • we would run a different kind of a percentage of a gel

  • than we would if we had very large proteins.

  • Student: Do they vary the actual size

  • of the plate itself, though?

  • Kevin Ahern: Do they vary the actual size of the plate itself?

  • Yeah, you can do that.

  • If you're looking at something that is quick and dirty,

  • you can run a very tiny little gel.

  • If you want to go and do 2D gel electrophoresis,

  • you would typically run a fairly large one

  • because you want to get as much

  • separation as possible for those.

  • So, yeah, they do vary the size, as well.

  • Okay.

  • So, good questions.

  • That was pretty much what I wanted to say.

  • I didn't show this last time,

  • but that was from a 2D gel electrophoresis,

  • the difference between a normal,

  • proteins from a normal human colon cell

  • and those from a colorectal tumor.

  • And you could find many differences between these,

  • and those are, as you might imagine,

  • of a considerable amount of interest.

  • Well, one of the things that we do,

  • we're doing all these techniques for,

  • is so that we can purify it, that is,

  • so that we can make plenty

  • of a pure amount of compound of proteins.

  • And, as I mentioned earlier,

  • one of the things we see in the purification process

  • is that that purification,

  • we never really know what's going to work before we do it.

  • So we have to really be very careful to monitor

  • during the purification process where is my protein.

  • Is it in the pellet?

  • Is it in the liquid?

  • Is it in the first part that comes off the anion exchange

  • or the last part that comes off of the anion exchange?

  • Because the worst thing that you can do is assume

  • it's in one fraction and throw that fraction away.

  • And I can tell you hundreds of stories

  • of people who've done exactly that.

  • So people get very careful.

  • If you've been working on this,

  • this is your PhD project, or whatever,

  • you're going to be checking every component

  • of that purification process to make sure

  • that you're not throwing the baby out with the bath water,

  • as it were.

  • Yes, sir?

  • Student: So I assume some of your stories are like,

  • okay, the protein should be here...

  • Kevin Ahern: Should be here, yeah.

  • Student:...but it's actually in this part.

  • Kevin Ahern: Right.

  • Right.

  • So that can be a real problem if that protein

  • is life and death for you,

  • in terms of a thesis or something.

  • So what you see here on the screen

  • is a sort of a depiction of protein separation

  • of a purification process.

  • Here is the unpurified.

  • Here was the stuff after we did one fractionation.

  • I haven't talked about salt fractionation,

  • but one way of separating things.

  • Another separation.

  • Another separation.

  • At each step, it's getting purer and purer,

  • until we finally see at the end,

  • hopefully, something that is almost absolutely pure

  • for us to work with.

  • Well, there are some considerations for that,

  • relevant to the numbers,

  • and I need to sort of step you through this

  • and tell you a little bit about this.

  • So one of the things we want to know in doing a purification is,

  • first of all, where is our protein?

  • But, second, how efficiently am I purifying this protein?

  • Because I'm going to be publishing this result,

  • and I want to report to others,

  • "Hey, this method really is good.

  • "It works very well,"

  • et cetera, so that others will have an idea about

  • how much material they're going to get out of it.

  • And so what you see on the screen

  • is a table following the purification of a protein.

  • And, yes, I think that you should be able to do calculations

  • like I'm going to describe to you in a second here.

  • So what we see are the several steps in this purification

  • that you saw in the last figure.

  • You had a homogenization just to bust open the cells.

  • You fractionated it.

  • You did ion exchange chromatography.

  • You did gel filtration.

  • And finally, you did affinity chromatography.

  • And so what this table is showing you is,

  • really, how much of the protein that you're getting,

  • apart from everything else in the process.

  • So, in this case, we started with a protein

  • and we had 15,000 milligrams of protein.

  • And it's very easy to determine

  • the protein concentration of a sample.

  • I bust open a bunch of cells.

  • Maybe I drew up a liter of cells.

  • I bust open the cells,

  • and I get 15,000 milligrams,

  • which is quite a bit of protein,

  • out of my cells, that's here.

  • I haven't done any purification.

  • All I've done is just bust open the cells.

  • So I need to know how much material I have to start with.

  • Well, that's a good starting point,

  • but of more importance to me is,

  • well, how much of my protein is in there?

  • And I don't know in terms of weight

  • but I can measure the activity of the protein.

  • Let's imagine my protein converts one molecule into another.

  • I can take that crude mix and take a very tiny aliquot of it

  • and treat it with this compound that it normally acts on.

  • And I can ask the question,

  • well, how many molecules of this compound

  • that it works on got converted?

  • So I have a definition there of what's called a "unit."

  • Let's say a unit might be a conversion of one nanomole

  • of this molecule into something else.

  • So I would say, well, I measure how many units I have

  • in my total mix that's there.

  • In this case, I had 150,000 units of my desired protein.

  • The specific activity of that is the total

  • number of units that I have,

  • divided by the total amount of protein that I had.

  • So specific activity would give me units per milligram.

  • This would be 150,000 divided by 15,000, or 10.

  • The yield is 100%.

  • And the yield is 100% because this is my starting material.

  • I haven't done anything to it.

  • I haven't lost anything.

  • I haven't gained anything.

  • And my purification level is 1 because,

  • again, I haven't purified,

  • I haven't done anything with it.

  • This is just where I started at.

  • After the salt fractionation,

  • I go back and I look and say,

  • "Whoa!

  • I lost a lot of protein here."

  • I've only got 4,600 milligrams of protein,

  • buthoa!悠 still have most of my protein activity.

  • That's really good.

  • I've retained a good deal of my original.

  • I only lost 12,000 units,

  • but I got rid of about 2/3 or 3/4 of the total protein.

  • So this tells me that I have

  • purified my protein to some extent,

  • because now my specific activity has changed

  • from 10 units per milligram to 30 units per milligram,

  • meaning I got rid of a bunch of junk I didn't want.

  • I threw out a little bit of the baby with the bath water,

  • maybe the leg or something like that.

  • [scattered laughter]

  • But... that's bad.

  • [scattered laughter]

  • Bad professor, okay?

  • But I have 30 units per milligram.

  • My yield is the number of units that I have now,

  • compared to how many units I started with.

  • So this, divided by this, times 100 gives me,

  • I've got 92% yield.

  • That's pretty darn good.

  • Ninety-two percent of my protein is there,

  • and three-quarters of the junk I don't want is gone.

  • The purification level is 3 because the specific activity

  • improved by a factor of 3,

  • 30 divided by 10 gave me 3.

  • Well, I can continue this process,

  • and you can go through the numbers

  • and see each of these as you go through.

  • And the bottom line will be that as I get

  • further and further to the bottom,

  • I keep losing more and more of my protein.

  • There's no method that's going to be absolute

  • in terms of keeping all my protein.

  • But what we see is that the specific activity goes up enormously,

  • which means that this stuff right here is really relatively full

  • of my protein and there's very little other stuff that's there.

  • So this has a total activity, 52,500 units,

  • and it only has 1.75 milligrams of protein.

  • That's 30,000 units per milligram.

  • I've got a yield of 35%,

  • meaning I did throw away a lot of the protein.

  • But, by golly, I purified that sucker by a factor of 3,000.

  • Alright?

  • So I got rid of an awful lot of junk in the methods that I used

  • to purify my protein.

  • You might say, "Well, how do you know when

  • you get it absolutely pure?"

  • And the answer is, you never really know.

  • But you can analyze it on a gel and you can see,

  • are there a bunch of other bands on this gel?

  • Or is it just my protein that I'm seeing on that gel?

  • And that can be a really useful thing.

  • If we look at the gel that I just showed you,

  • Oh, wrong one,

  • okay, we can see this has basically gone,

  • they don't really show other bands here unfortunately,

  • but you might imagine that if you had a protein

  • that wasn't very pure you would see some other molecular sizes

  • that would be in this particular lane.

  • And you would see those disappear the further

  • I get along with my purification.

  • Questions on what I've just told you?

  • Yes, sir?

  • Student: So the purification level was the current specific

  • activity level divided by the previous?

  • Kevin Ahern: The purification level is the current

  • specific activity level divided by the starting level.

  • So the starting had 10,

  • and I'm looking at how much I've purified it compared

  • to what it was I started with.

  • So in this case, I had 10 units per milligram to start.

  • Down here, I had 30,000 units per milligram.

  • So my purification level is 30,000 divided by 10, or 3,000.

  • Make sense?

  • Yes?

  • Student: So the gel filtration is the SDS-PAGE?

  • Kevin Ahern: No.

  • Gel filtration is the method I talked about the other day

  • where you separate on the basis of size.

  • Student: I thought SDS-PAGE separated by size.

  • Kevin Ahern: It does.

  • But there are other things that separate by size.

  • So gel electro-, that's where you had the beads

  • with the little holes in them?

  • Yeah.

  • That's gel filtration.

  • Yes, sir?

  • Student: If you continued purifying this,

  • would you start seeing diminishing returns?

  • Kevin Ahern: If you kept purifying this,

  • would you see diminishing returns?

  • In fact, at every step of purification

  • you will always see diminishing returns,

  • yes.

  • Yeah.

  • Alright.

  • So that's purification.

  • What I want to do is spend a little bit of time

  • talking about characterization of proteins,

  • and we're going to talk about some techniques of spectroscopy

  • and also some just simple tools to work with proteins.

  • I'm going to skip over amino acid analysis

  • and I'm not going to hold you responsible for it.

  • There are, suffice it to say that there are a variety

  • of chemical and chromatographic tools for analyzing

  • the amino acids in proteins,

  • but the reality is that people

  • don't do that very much anymore

  • because it's much easier

  • to determine the amino acid composition

  • of a protein if you have the DNA sequence,

  • and it's much easier to sequence the DNA.

  • So I'm not going to talk about amino acid analysis,

  • and as I said, you won't be responsible for that.

  • One of the things that we have to do in working with proteins

  • let's say we get our protein very pure

  • and we want to start to characterize it,

  • start to understand better what all is in it,

  • what it's comprised of,

  • one of the things that we have to do

  • is we actually have to take and cut

  • the protein into smaller pieces.

  • Some proteins can be quite large,

  • many have molecular weights of 200,000 or more,

  • and those are really very difficult for us to work with

  • some of the methods I'm going to be showing you about.

  • So it's desirable, then, to be able to cut proteins

  • into smaller pieces.

  • And to do this, we use a series of chemical reagents or enzymes,

  • depending on what we're trying to do,

  • that will specifically break peptide bonds

  • in a protein at specific places.

  • The first one I'll tell you about is actually a chemical reagent.

  • It's called cyanogen bromide.

  • Now, cyanogen bromide has a very interesting and useful property.

  • When you take and you treat a protein with cyanogen bromide,

  • what happens is every place that there's a methionine residue

  • the peptide bond will be broken.

  • Every place there's a methionine,

  • the peptide bond will be broken.

  • Well, since methionines occur in any given protein

  • at specific places, we get a specific set of fragments

  • that arise from treatment of a protein with cyanogen bromide.

  • You can see other reagents that are here.

  • Blah, blah, blah.

  • The only chemical reagent that I expect that

  • you will know about is cyanogen bromide.

  • It's the most commonly used one,

  • and it is very simple,

  • in terms of what it does.

  • In addition to chemical reagents that we use

  • to chop proteins into smaller pieces,

  • we commonly use enzymes.

  • And enzymes come from our digestive system,

  • for the most part, okay?

  • Our body has enzymes that break down proteins

  • in our digestive system so that when we eat food

  • and there are proteins in there,

  • we can break those proteins down into amino acids

  • that we can use for our own purposes.

  • So some of these that we use are really useful.

  • One is trypsin.

  • Trypsin is a very simple one to understand.

  • It cuts on the carboxyl side,

  • and I'll show you a figure for this in a second

  • but it cuts on the carboxyl side

  • of lysine and arginine residues.

  • That's really useful.

  • So again, lysine and arginines are at specific places

  • in a given protein.

  • By treating a protein with trypsin,

  • I get a specific set of fragments arising from that cleavage.

  • We'll talk more about trypsin later in the term.

  • Thrombin cleaves on the carboxyl side of arginine.

  • That's a very useful tool.

  • So if I compared the pattern that arose

  • from cutting a protein with thrombin

  • compared to the cutting with trypsin,

  • I would guess I would probably get more fragments with trypsin

  • because trypsin cuts near two different amino acids

  • whereas thrombin only cuts near arginine right here.

  • Well, later in the term I'll talk about chymotrypsin.

  • I'll just point out that it cuts near all of these.

  • But you'll notice a pattern.

  • This is a benzene ring, a benzene ring, a benzene ring.

  • So these guys all have aromatic amino acids

  • that they cleave next to, right here.

  • In addition, to some extent it will cleave other amino acids.

  • And the one characteristic you could notice of all of these,

  • at least of four of the five,

  • is that they are hydrophobic.

  • Tyrosine's the only one that's not very hydrophobic there.

  • Well, again, I do think that you should know where thrombin cuts.

  • I think you should know where trypsin cuts.

  • And I think you should know where cyanogen bromide cuts.

  • The other ones are just sort of,

  • I don't think it's really necessary for our purposes.

  • But you should know that enzymes are very useful.

  • They're called proteolytic enzymes,

  • or they're called proteases.

  • Proteases cleave peptide bonds in other proteins.

  • And if the question is, will they cleave bonds within themselves,

  • the answer is, one protease can cleave another protease, you bet.

  • And so, if you leave a protease in a tube over a period of time,

  • it will eventually lose all of its activity

  • because it cuts itself to pieces.

  • Student: Well, how do you store it, then?

  • Kevin Ahern: How do you store it?

  • You store it frozen.

  • Student: Oh.

  • Kevin Ahern: Yep.

  • So you isolate it under conditions where it's not very active,

  • and then you ship it and keep it's frozen

  • so that it's not able to act.

  • Shannon?

  • Student: Is it true that you can store it refrigerated if it's dry?

  • Kevin Ahern: Can you refrigerate it if it's dry?

  • It depends on the protease, but yeah,

  • some of them are actually shipped dry,

  • but they're often kept frozen for that very same reason, as well.

  • Let's see.

  • I talked very briefly before about reducing disulfide bonds.

  • I'll just very briefly show you here, again.

  • I mentioned that, when I talked about mercaptoethanol,

  • I said mercaptoethanol would take a disulfide bond

  • and convert it back to sulfhydryl groups.

  • I said, at the time,

  • there's a molecule called dithiothreitol that will do the thing,

  • and now you see dithiothreitol doing the same thing.

  • What's happening is that this guy is donating electrons

  • to this disulfide bond.

  • So the electrons go here.

  • And when this guy loses its electrons,

  • it becomes a disulfide bond.

  • The same thing happens with mercaptoethanol,

  • actually, as it's reducing disulfides in a protein.

  • This can be important because,

  • again, if we want to get the pieces apart,

  • we may want to break the disulfide bonds of a protein.

  • And, let's see.

  • No surprise.

  • You've had in basic biology,

  • the relationship of the genetic code,

  • which shows how the sequence of DNA ultimately can be converted

  • into the sequence of amino acids in a protein.

  • And, as I mentioned earlier,

  • it's actually much easier to determine

  • the sequence of a protein by sequencing its DNA than

  • to try to determine the individual sequence of amino acids

  • solely starting from the protein alone.

  • So DNA sequence is a much easier way

  • to determine your protein sequence.

  • Well, I want to spend some time talking about

  • an immunological technique that allows us to identify

  • specific proteins from an SDS gel.

  • So let's imagine I've got that SDS get that

  • I separated those proteins on before.

  • And you saw there were a series of bands that were there,

  • on the side of the gel.

  • One of the questions you might ask is,

  • "Well, I'm really interested in a particular protein of my own.

  • How could I tell which one of those bands

  • is the one I'm interested in?"

  • So one way of doing that is this technique that's called

  • "western blotting."

  • And let me take a few minutes

  • and describe western blotting to you.

  • To do that, I need to, first of all,

  • tell you a little bit about antibodies.

  • Antibodies are proteins of the immune system

  • that provide protection for us against outside invaders.

  • And they work by binding to specific structures.

  • So this is a schematic diagram of an antibody.

  • It has one end that binds.

  • It actually has two ends that bind to specific structures.

  • And I'll describe those structures in a second.

  • And the immune system,

  • this allows the immune system to fight off invaders.

  • We'll talk about the immune system in 451 next term.

  • But this allows the immune system to fight off invaders.

  • The reason that we use antibodies

  • in this technique is because of their ability to bind

  • to only very specific structures.

  • So let's imagine that I'm interested in studying a protein

  • that's a protein from HIV.

  • And I've got this mixture of proteins on the side of my gel,

  • and I really wanted to know which protein

  • there was the one that was mine.

  • To do this, I would have had to made an antibody

  • against my protein of interest.

  • So let's say I've got my purified protein.

  • I'm interested in studying it,

  • using a western blot technique.

  • I would actually inject this protein into, say, a bunny rabbit

  • or something like that.

  • And the bunny rabbit's immune system would see this

  • protein coming in as a foreign invader.

  • It doesn't hurt the bunny rabbit in any way.

  • The bunny rabbit's immune system makes antibodies against that.

  • And then I can collect some blood from the bunny rabbit

  • and isolate those antibodies that bind to my protein.

  • And that can take a few weeks to get set up.

  • Just a second, Shannon.

  • I've got my antibody that's there.

  • And it's binding specifically to my protein.

  • Did you have a question?

  • Student: Oh, yeah.

  • Is it possible to carry this out in vitro?

  • Kevin Ahern: To carry out the antibody generation in vitro?

  • Student: Yeah.

  • Kevin Ahern: No.

  • The immune system has to recognize it

  • and then the antibody has to be synthesized.

  • So it can't be made in vitro, no.

  • Alright.

  • Now, so I've got an antibody that binds to my protein,

  • alright?

  • That's the most important component

  • of what I'm going to be showing you.

  • And the beauty of an antibody is it's specific.

  • It will bind to my protein but it won't bind

  • to all the other proteins I might find in blood,

  • or all the other proteins I might find in a plant cell

  • or whatever it is that I'm putting on that gel.

  • It will only bind to the protein that I'm interested in, ideally.

  • So, I've got an antibody that's specific for my,

  • it's called the "antigen."

  • That's the thing it binds to.

  • So there's the antibody.

  • There's the antigen.

  • There's the binding.

  • And the binding can be quite tight,

  • and is necessary for us to do our analysis.

  • Now, this antibody is going to be used in this technique called

  • western blotting that I'm going to show you.

  • So I've got my mixture of proteins.

  • I take my mixture of proteins

  • and I apply them to the top of an agarose gel,

  • I'm sorry, a polyacrylamide gel.

  • And I separate them.

  • So I've got an SDS-PAGE.

  • I've separated my proteins.

  • In this case, they've actually cut out the specific band,

  • but you could do the whole gel, if you wanted to.

  • And I take those proteins in that gel.

  • Gels are kind of hard to handle.

  • They fall apart real readily,

  • like working with Jello or something.

  • So I can take and I can actually use an electric current

  • to transfer those proteins onto a membrane,

  • Onto a membrane, like a sheet of,

  • sometimes you can use like a specialized sheet of paper.

  • So you would transfer all the proteins

  • that's on there onto this sheet of paper.

  • And the proteins would then be stuck to that paper.

  • And we can use techniques to make them stick quite strongly.

  • So now I've got my paper that was the exact match of my gel,

  • and it's got those proteins attached to it.

  • I take the paper and I transfer it to a bag that

  • has some buffer in it,

  • and I add my antibody.

  • I add my antibody.

  • The antibody is given some time,

  • a few hours, to bind to what it's going to bind to.

  • Let's say my protein is right there.

  • The other ones aren't proteins of interest.

  • The antibody binds and I take the piece of paper out.

  • I wash it so that all the things that aren't bound

  • specifically to my protein,

  • all the other antibodies,

  • come off.

  • And then I use a reagent that basically tells me,

  • I ask the question, "Where are the antibodies?"

  • So there are reagents that will light up color

  • where there's an antibody.

  • And now, by identifying the color, I can say,

  • "There's my protein."

  • And I can go all the way back here and say,

  • "There's where my protein was."

  • So it's a very useful technique for specifically identifying

  • a protein of interest in a mixture of other proteins.

  • Very, very useful.

  • A very important technique for me to be able to identify

  • a protein after I've done an SDS-PAGE.

  • So I'm going kind of fast there.

  • I'll slow down and take any questions you might have.

  • Yes.

  • Back in the back.

  • Student: So does the protein, like,

  • do all the other proteins come off of that?

  • Kevin Ahern: Do all the other proteins come off?

  • No, they don't.

  • But the antibody doesn't bind to them,

  • so when I treat to find antibodies,

  • only the one that's got antibodies on it will light up.

  • Yes, sir?

  • Student: Could you treat those antibodies beforehand.

  • Kevin Ahern: So his question was,

  • could I treat the antibodies beforehand?

  • And the answer is, yes, I could.

  • Sometimes people will take,

  • and it's actually easy to put a color onto an antibody,

  • so I don't have to do the treatment afterwards.

  • So now I can just look again,

  • and say, "Where's the color?"

  • There's a variety of ways of visualizing this thing right here.

  • Student: Can you assay the amount?

  • Kevin Ahern: Can you assay the amount?

  • Western blotting gives you a rough idea of amount,

  • but it's not real good for overall quantitation.

  • But it gives you a ballpark idea of the amount,

  • yes.

  • Yes, sir?

  • Student: Okay.

  • You've identified which one out of your original SDS-PAGE

  • is a protein of interest.

  • Kevin Ahern: Yep.

  • Student: Is there a way to dissolve away the gel?

  • Or to isolate the protein for experimental use?

  • Kevin Ahern: Oh, very good question.

  • So he says I've identified my protein.

  • Is there a way I can recover that protein and use it?

  • The answer is, you can extract a protein from there,

  • but frequently you won't want to do that.

  • Anybody know why you wouldn't want to do that?

  • Student: You've denatured the protein.

  • Kevin Ahern: You've already denatured the protein.

  • So what's in there is already probably not of any use to you

  • if you're interested in a protein that's active.

  • Yes, you can,

  • and commonly what people will do is take that

  • and analyze it in another way.

  • So they'll cut it out and use a technique of spectroscopy

  • to further identify it.

  • So the answer is, yes, you can,

  • but it depends on what you want to do with it

  • in terms of whether that's going to be practical for you or not.

  • Student: But you could recover it through like

  • crystalize for an X-ray exam?

  • Kevin Ahern: You would not take this

  • and use it for crystallography

  • or for other analysis like that, no.

  • No.

  • It wouldn't be of use to you.

  • But it is of use in other ways.

  • Yes?

  • Student: So after you identify it and know where it is on there

  • if you want to get more of it without denaturing it

  • Kevin Ahern: You probably didn't put your whole sample on here.

  • So you probably took a pretty small fraction.

  • Student: And then you just do it again

  • and you don't denature it the next time?

  • Kevin Ahern: Uh, no.

  • Because it's not denaturing it.

  • You don't know where it's going to go, right?

  • So there's other things that you have to do.

  • I'm just showing you one way of doing that isolation separation.

  • Yes, sir?

  • Student: You talk about developing antibodies in rabbits.

  • Does that work for non-animal proteins?

  • Kevin Ahern: Can I葉he question,

  • I think, is, will a rabbit make antibodies against

  • a non-animal protein?

  • The answer is, yes.

  • They are specific for structures.

  • So they'll work on proteins.

  • You can make 'em against DNA.

  • You can make 'em against RNA.

  • You can make 'em against carbohydrate,

  • So it's structure that's the important thing,

  • not the source of the molecule

  • or even the type of molecule that it is.

  • Good question.

  • Yes?

  • Student: We were talking the other day about prions.

  • Kevin Ahern: Uh-huh.

  • Student: And you said that immune systems

  • generally don't recognize them.

  • What if you did that cross-speciesation

  • and you injected that like into a different species

  • and it recognized it as a non-native...

  • Kevin Ahern: Okay, so, yeah.

  • The question is, that's getting a little involved,

  • but basically his question is,

  • can I make an immune system recognize a prion.

  • The answer is, yes I can.

  • But that doesn't mean it's going to be an effective treatment,

  • and that is what we were talking about

  • with the immune system the other day.

  • So, yes, I can make antibodies against that,

  • but that's not necessarily going to be something

  • that's going to be useful in terms

  • of helping to treat the disease.

  • In other words, my immune system is not going to protect me

  • against that protein.

  • So... yeah.

  • Ah-bah-dah.

  • Let's see here.

  • So let's spend just a couple of minutes

  • I'm not too far from finishing stuff here

  • spend a couple of minutes talking about,

  • you asked the question earlier about,

  • can I pull this protein out of this gel and use it for something?

  • And I can.

  • So let's imagine I've taken and I've identified my band.

  • I have cut out that band.

  • It could either be from a gel,

  • like I showed here,

  • or it could be from a 2D gel.

  • Either way, I could pull out the band of interest and say,

  • "Okay.

  • Here is my protein.

  • I've separated it by gel electrophoresis.

  • I'd like to know,

  • what is it?

  • What's the sequence of it, for example?"

  • Well, I don't know where in the DNA it came from.

  • I have to actually sequence,

  • in this case, the protein itself.

  • So I take this purified protein that I've got

  • and I might treat it with some enzymes

  • to break it into smaller pieces.

  • I might treat it with a chemical like cyanogen bromide

  • to break it into smaller pieces.

  • And breaking it into smaller pieces is going to be important

  • because the next technique I'm going to describe to you

  • works very well on relatively small pieces of polypeptides.

  • This technique is called MALDI-TOF.

  • And I'm going to show you the image first,

  • okay?

  • Oh, blast it.

  • I'll start with this.

  • MALDI-TOF is a,

  • MALDI, M-A-L-D-I,

  • stands for "matrix assisted laser desorption ionization."

  • You don't need to know that.

  • Okay?

  • It's a mouthful.

  • I always have to look it up each time,

  • so I remember what it is.

  • Matrix assisted laser disruption ionization.

  • What does that mean?

  • It means this technique uses a laser

  • to make a sample volatilize.

  • That's the first part of what happens in mass spec.

  • How many people have done mass spec in a chemistry lab?

  • So mass spec tells us mass.

  • And mass specs work in vacuum chambers.

  • And ions in the vacuum chamber get accelerated

  • and they get accelerated to move up to a detector

  • which detects when things hit them.

  • So MALDI-TOF is a specialized form of mass spectrometry

  • that allows me to analyze relatively large molecules,

  • like polypeptides.

  • To do MALDI-TOF, one takes a protein sample that's purified,

  • that little band that I had,

  • and mixes it with some material

  • that makes it form a sort of a crystal.

  • And that would be on the end of a,

  • let's say a pinhead that I could put into a chamber

  • that would be evacuated.

  • So I've got my sample that's on a pinhead.

  • I put it in this evacuated chamber and it's sitting there,

  • waiting for the analytical process to begin.

  • The "L" said "laser,"

  • and laser plays an important role in this process.

  • The laser,

  • I thought I had that figure here and I just don't see it.

  • Okay.

  • Well, it's in the book.

  • I'll post the figure later,

  • to show you.

  • The laser is, there's a laser that's pointed,

  • and the laser hits that sample in the evacuated chamber.

  • So the evacuated chamber has my crystallized material.

  • When the laser hits it,

  • that crystallized sample volatilizes,

  • meaning it leaves the pinhead and goes into a gaseous phase.

  • In the process of that happening,

  • my sample becomes ionized.

  • It becomes charged.

  • Okay?

  • Because it's charged, an electric field will attract

  • it to the detector at the other end.

  • Okay?

  • Let's imagine I've got a sample that's got,

  • let's say, two things in it.

  • One that has a molecule with a mass of 500

  • and one that has a mass of 1,000.

  • And they're both charged +1.

  • Which one's going to make it faster through the chamber?

  • The 500, right?

  • Because it's got the same charge.

  • It's got less mass.

  • It's going to have less inertia,

  • and it's going to be accelerated faster

  • and is going to arrive at the detector faster.

  • It means that, if we measure carefully the time it takes

  • from volatilization over to the time the detector detects it,

  • that time interval actually tells us the size.

  • Because it'll take something twice as long that's 1,000

  • in molecular weight as it will for something that's 500.

  • That's really useful for us

  • because with that technique we can determine molecular masses

  • with amazing efficiency.

  • Because of that, we can take a sample that has a polypeptide,

  • and that polypeptide, when it ionizes, will break into pieces.

  • The places where it will break are peptide bonds.

  • So here's a full-length polypeptide that's there.

  • When it ionizes, some of them will be broken here,

  • and I'll get one amino acid.

  • Some will be here, I'll get two amino acids.

  • Some will be broken here, I'll get three amino acids.

  • And if I determine the masses of all those

  • the difference between the peaks is the difference

  • of each amino acid that comes off

  • I can actually determine the sequence of the thing I started with.

  • Yes, it's complicated and yes,

  • it takes a computer to do it.

  • I'm not going to sit and stare at it, myself.

  • But the beauty is that, in a single mass spectrometric analysis,

  • I can determine the sequence of amino acids of that polypeptide

  • I put into the chamber.

  • Now that is really powerful.

  • Because of this technology,

  • because of this technology,

  • a scientist in a modern mass spec lab can determine

  • the sequence and, thus,

  • the identity of 4,000 proteins a day.

  • We could take all the proteins that was on a 2D gel,

  • cut out each spot, have each one analyzed,

  • have each one identified, in a single day.

  • That is an incredibly powerful technique.

  • Question?

  • Student: How long have they be able to do that?

  • How old is this technology?

  • Kevin Ahern: How what?

  • Student: How old is this technology?

  • Kevin Ahern: This technology dates to the '90s.

  • Yeah.

  • So it's relatively new.

  • Yes?

  • Student: So why do you,

  • how do you know which end it starts at,

  • so you have the...

  • Kevin Ahern: Okay.

  • So there are at both ends,

  • and you can actually get this one, as well.

  • That's what that computer has to sort out.

  • Okay?

  • Okay.

  • Kevin Ahern: A lot of stuff there.

  • Let's call it a day and I will see you on Wednesday.

  • Student: [inaudible]

  • Kevin Ahern: I'm sorry?

  • Student: Will we get to talk about photo [inaudible].

  • Kevin Ahern: We're not going to talk about [inaudible].

  • Sorry.

  • Yeah.

  • [END]

Another Monday beckons, another week beckons.

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