WEBVTT 00:00:16.490 --> 00:00:19.170 PROFESSOR: All right, so fluorescence. 00:00:19.170 --> 00:00:21.270 You know, I know you hear this from me a lot. 00:00:21.270 --> 00:00:24.570 But this really is my favorite topic. 00:00:24.570 --> 00:00:27.990 The applications of luminescence and fluorescence 00:00:27.990 --> 00:00:32.650 in service to biology are incredibly important. 00:00:32.650 --> 00:00:35.820 So what I'm going to try to do in these two lectures 00:00:35.820 --> 00:00:39.570 is explain to you the difference between fluorophores 00:00:39.570 --> 00:00:44.220 that we can encode into proteins through genetic engineering 00:00:44.220 --> 00:00:46.440 and fluorophores that we use that are 00:00:46.440 --> 00:00:50.340 made by chemists in the lab but then appended to molecules. 00:00:50.340 --> 00:00:54.480 So today we'll talk about the nuts and bolts of fluorescence. 00:00:54.480 --> 00:00:56.100 And then on Wednesday, we'll start 00:00:56.100 --> 00:01:01.740 to see some of these tools that you've seen images of. 00:01:01.740 --> 00:01:05.640 We love to wow you with images of fluorescent cells and cells 00:01:05.640 --> 00:01:06.480 in action. 00:01:06.480 --> 00:01:09.510 But I want to step back and actually show you how 00:01:09.510 --> 00:01:11.280 that all came about. 00:01:11.280 --> 00:01:13.900 Where do these fluorescent proteins come from? 00:01:13.900 --> 00:01:15.300 What are we looking for? 00:01:15.300 --> 00:01:18.090 How much protein engineering was done 00:01:18.090 --> 00:01:22.470 to make these such an amazingly useful set of molecules, 00:01:22.470 --> 00:01:26.280 macromolecules to really allow us in real time 00:01:26.280 --> 00:01:28.180 to study biology? 00:01:28.180 --> 00:01:31.630 And there are many, many other applications as well. 00:01:31.630 --> 00:01:35.130 So we're going to talk about luminescence and fluorescence 00:01:35.130 --> 00:01:36.450 in general. 00:01:36.450 --> 00:01:38.415 Luminescence is the general term. 00:01:43.830 --> 00:01:47.350 And fluorescence is a little bit more specific. 00:01:47.350 --> 00:01:49.660 There are different types of luminescence. 00:01:49.660 --> 00:01:51.910 And you'll get to see some of those varieties 00:01:51.910 --> 00:01:53.080 of luminescence. 00:01:53.080 --> 00:01:56.660 I've put a decent amount of our content today on the screen. 00:01:56.660 --> 00:01:58.580 So we'll go up here and take a look. 00:01:58.580 --> 00:02:02.020 So luminescence in general is the emission 00:02:02.020 --> 00:02:05.290 of light not associated with heat, not 00:02:05.290 --> 00:02:09.190 like a burning flame which has a lot of light accompanying it. 00:02:09.190 --> 00:02:13.500 But rather the emission of light in the absence of heat. 00:02:13.500 --> 00:02:18.580 And there are different types of luminescence the biologists use 00:02:18.580 --> 00:02:21.850 intertwined into biological experiments 00:02:21.850 --> 00:02:24.100 to illuminate life, to understand 00:02:24.100 --> 00:02:26.710 details of cellular activity. 00:02:26.710 --> 00:02:30.040 But also as reagents in diagnostics 00:02:30.040 --> 00:02:33.340 in all kinds of imaging modalities. 00:02:33.340 --> 00:02:34.600 And through these two-- 00:02:34.600 --> 00:02:37.270 three lectures, actually, because Professor Martin 00:02:37.270 --> 00:02:39.970 will give you one that has even more imaging in it-- 00:02:39.970 --> 00:02:43.630 you'll sort of really get to understand where these pretty 00:02:43.630 --> 00:02:45.950 magical reagents come from. 00:02:45.950 --> 00:02:48.100 So the two types of luminescence that we 00:02:48.100 --> 00:02:53.200 won't discuss in detail today, first of all chemiluminescence. 00:02:53.200 --> 00:02:56.140 This is a molecule known as luminol 00:02:56.140 --> 00:02:57.610 sitting in a little bile. 00:02:57.610 --> 00:03:01.210 I think I like fluorescence so much because the images are so 00:03:01.210 --> 00:03:02.650 captivating. 00:03:02.650 --> 00:03:05.890 Now, things like luminol, has anyone heard of luminol before? 00:03:05.890 --> 00:03:09.430 Does anyone-- yeah, do you watch a lot of CSI or-- 00:03:09.430 --> 00:03:10.090 yeah. 00:03:10.090 --> 00:03:13.935 So tell everybody what people use luminol for. 00:03:13.935 --> 00:03:16.590 AUDIENCE: To pick up on a blood spatter or remnants of blood. 00:03:16.590 --> 00:03:20.170 PROFESSOR: Yeah, so when you see these TV 00:03:20.170 --> 00:03:24.580 shows and there's this beautifully clean motel room 00:03:24.580 --> 00:03:27.880 and nothing looks like it ever happened there. 00:03:27.880 --> 00:03:30.880 But people thought there was a murder took place there, 00:03:30.880 --> 00:03:33.790 you'll notice they come in with these spray bottles. 00:03:33.790 --> 00:03:37.623 And spray all over the carpets, and the drapes, and the chairs. 00:03:37.623 --> 00:03:39.040 And then there's this great moment 00:03:39.040 --> 00:03:43.420 where they turn off the light and luminol interacts 00:03:43.420 --> 00:03:48.280 with the heme of blood at an amazingly sensitive level, 00:03:48.280 --> 00:03:50.650 such that when the lights are turned out, 00:03:50.650 --> 00:03:53.950 the room's just sort of this battlefield 00:03:53.950 --> 00:03:56.560 of bright luminescence that indicates 00:03:56.560 --> 00:03:58.130 that this was a crime scene. 00:03:58.130 --> 00:04:03.180 So that's the most famous luminol sort of example. 00:04:03.180 --> 00:04:05.690 So that is what would be called chemiluminescence, 00:04:05.690 --> 00:04:08.650 the interaction of a chemical with another chemical 00:04:08.650 --> 00:04:10.420 to give luminescence. 00:04:10.420 --> 00:04:12.900 Another pretty useful type of luminescence 00:04:12.900 --> 00:04:14.890 is bioluminescence. 00:04:14.890 --> 00:04:16.959 I've done a lot of scuba diving in my life. 00:04:16.959 --> 00:04:20.290 And there's nothing more exciting than a dive at night 00:04:20.290 --> 00:04:24.220 where the whole ocean is this sort of inky black. 00:04:24.220 --> 00:04:26.080 And sometimes you'll move your arms 00:04:26.080 --> 00:04:28.450 through the black water at night. 00:04:28.450 --> 00:04:31.470 And you'll see all these, like, little fireworks. 00:04:31.470 --> 00:04:35.530 And many, many marine organisms actually 00:04:35.530 --> 00:04:37.600 undergo bioluminescence. 00:04:37.600 --> 00:04:41.170 It's a biological reaction that causes luminescence. 00:04:41.170 --> 00:04:44.110 And this is a cuttlefish shown here 00:04:44.110 --> 00:04:47.560 in this image in the corner where 00:04:47.560 --> 00:04:49.550 they are brightly lit at night. 00:04:49.550 --> 00:04:51.340 And actually, it's just a whole party 00:04:51.340 --> 00:04:54.820 there at night in the ocean where all sorts of organisms 00:04:54.820 --> 00:04:58.870 are signaling to other organisms through bioluminescence 00:04:58.870 --> 00:05:01.850 and reactions such as luciferase reactions. 00:05:01.850 --> 00:05:04.870 So in bioluminescence, a molecule of ATP 00:05:04.870 --> 00:05:09.130 is generally used and combined with another molecule 00:05:09.130 --> 00:05:11.680 through the action of an enzyme that ends up 00:05:11.680 --> 00:05:13.700 kicking out light energy. 00:05:13.700 --> 00:05:14.960 So those are both important. 00:05:14.960 --> 00:05:17.740 But what we're going to talk about principally 00:05:17.740 --> 00:05:19.690 is fluorescence. 00:05:19.690 --> 00:05:21.455 This is a more specific term. 00:05:26.370 --> 00:05:28.180 And you may wonder why I'm putting 00:05:28.180 --> 00:05:31.158 this in capital letters. 00:05:31.158 --> 00:05:32.950 The first thing to learn about fluorescence 00:05:32.950 --> 00:05:35.560 is how to spell fluorescence. 00:05:35.560 --> 00:05:37.480 So if you look at the word fluorescence 00:05:37.480 --> 00:05:41.950 and the first word first part of the word looks like flower, 00:05:41.950 --> 00:05:44.620 you know the stuff you bake your pumpkin pie with, 00:05:44.620 --> 00:05:46.270 you spelled it wrong. 00:05:46.270 --> 00:05:50.722 It actually is fluor, F-L-U-O-R-E-S. 00:05:50.722 --> 00:05:52.180 What are you guys whispering about? 00:05:52.180 --> 00:05:53.530 AUDIENCE: [INAUDIBLE]. 00:05:53.530 --> 00:05:55.280 PROFESSOR: Did I get something else wrong? 00:05:55.280 --> 00:05:56.471 AUDIENCE: The E-S. 00:05:56.471 --> 00:05:59.420 PROFESSOR: E-S, yeah, well, forget about that part. 00:05:59.420 --> 00:06:02.550 E-S. There's another C in there. 00:06:02.550 --> 00:06:04.520 There we go, we snuggled that in. 00:06:04.520 --> 00:06:07.140 So the important part is the first part. 00:06:07.140 --> 00:06:09.770 So make it look like you know what you're talking about 00:06:09.770 --> 00:06:11.390 and spell fluorescence correctly. 00:06:11.390 --> 00:06:14.810 I cannot tell you how many papers, 00:06:14.810 --> 00:06:17.340 scientific papers I read where they spelled fluorescence 00:06:17.340 --> 00:06:17.840 wrong. 00:06:17.840 --> 00:06:19.340 It's really hysterical. 00:06:19.340 --> 00:06:20.240 It's one of those-- 00:06:20.240 --> 00:06:23.300 there's two or three amazingly common typos 00:06:23.300 --> 00:06:24.440 in people's slides. 00:06:24.440 --> 00:06:26.750 One of them is spelling fluorescence wrong 00:06:26.750 --> 00:06:29.870 and the other one is spelling complement wrong. 00:06:29.870 --> 00:06:31.610 Complement as opposed to compliment 00:06:31.610 --> 00:06:33.530 where you're telling someone they look good. 00:06:33.530 --> 00:06:34.970 And complement where you're trying 00:06:34.970 --> 00:06:37.710 to sort of match up things. 00:06:37.710 --> 00:06:40.250 Anyway, but fluorescence is a key point. 00:06:40.250 --> 00:06:41.870 So what is fluorescence? 00:06:41.870 --> 00:06:58.030 It's the absorption of light energy by a molecule. 00:06:58.030 --> 00:07:01.330 Now it could be a small, organic molecule. 00:07:01.330 --> 00:07:05.020 It could be a small part of a fluorescent protein 00:07:05.020 --> 00:07:07.450 molecule that has a particular structure. 00:07:07.450 --> 00:07:11.350 But it will absorb light energy at a certain wavelength. 00:07:11.350 --> 00:07:15.070 So let's put this into just a cuvette experiment. 00:07:15.070 --> 00:07:19.090 These are the kinds of little containers 00:07:19.090 --> 00:07:21.470 that we use for certain types of fluorescence. 00:07:21.470 --> 00:07:24.220 We could use a plate and a plate reader. 00:07:24.220 --> 00:07:25.840 But this is quite common. 00:07:25.840 --> 00:07:28.170 So you shine light on this molecule. 00:07:32.090 --> 00:07:35.540 Do you want to nab the doors, the outer doors on both sides? 00:07:35.540 --> 00:07:36.150 I don't know. 00:07:36.150 --> 00:07:38.080 Everybody's pretty happy today. 00:07:38.080 --> 00:07:40.590 Anyway, and the molecule goes. 00:07:40.590 --> 00:07:45.430 And I'm going to just draw something, 00:07:45.430 --> 00:07:49.620 you know, with a bunch of double bonds and things. 00:07:49.620 --> 00:07:54.700 And the molecule absorbs light and goes to an excited state. 00:07:54.700 --> 00:08:00.300 So this is the ground state before light. 00:08:00.300 --> 00:08:09.070 After light you get the molecule in an excited state. 00:08:09.070 --> 00:08:13.540 So it has absorbed that light energy. 00:08:13.540 --> 00:08:16.980 So you've hit it with a wavelength of light. 00:08:16.980 --> 00:08:19.590 And I'm going to redefine all these terms properly 00:08:19.590 --> 00:08:20.700 in a moment. 00:08:20.700 --> 00:08:26.250 Lambda excitation of a particular wavelength. 00:08:26.250 --> 00:08:29.010 Once that molecule has absorbed light, 00:08:29.010 --> 00:08:33.750 there's a very transient period until the molecule lets out 00:08:33.750 --> 00:08:35.205 energy in the form of light. 00:08:42.669 --> 00:08:46.090 And returns back to its ground state now. 00:08:49.510 --> 00:08:52.510 So that the photo physics of fluorescence 00:08:52.510 --> 00:08:55.510 involves the excitation of a molecule with light 00:08:55.510 --> 00:08:56.980 of one energy. 00:08:56.980 --> 00:08:59.670 That light energy is at a particular wavelength 00:08:59.670 --> 00:09:03.910 so it's called its lambda of excitation. 00:09:03.910 --> 00:09:06.130 That molecule very, very transiently, 00:09:06.130 --> 00:09:08.620 it's usually picoseconds or nanoseconds 00:09:08.620 --> 00:09:10.240 for organic molecules. 00:09:10.240 --> 00:09:13.870 It may be a little bit longer for other types of complexes 00:09:13.870 --> 00:09:17.050 and may stretch to the microsecond or millisecond. 00:09:17.050 --> 00:09:19.750 But most of what we talk about will be picosecond 00:09:19.750 --> 00:09:21.730 to nanosecond lifetime. 00:09:21.730 --> 00:09:25.810 And once it's excited, it just drops its energy back out 00:09:25.810 --> 00:09:27.670 and goes back to the ground state. 00:09:27.670 --> 00:09:31.030 And the most important thing that you want to remember 00:09:31.030 --> 00:09:41.510 is the wavelength, which is given in nanometers. 00:09:41.510 --> 00:09:49.000 And the wavelength for emission, which is also in nanometers. 00:09:49.000 --> 00:09:51.200 This wavelength is higher energy. 00:09:55.110 --> 00:09:57.520 Obviously you don't create energy 00:09:57.520 --> 00:09:59.560 when you shine light on something. 00:09:59.560 --> 00:10:02.650 You'd be breaking a few fundamental rules if you did. 00:10:02.650 --> 00:10:07.870 So the light that comes back out is lower energy 00:10:07.870 --> 00:10:10.570 because there's been rearrangements in the excited 00:10:10.570 --> 00:10:11.750 state of the molecule. 00:10:11.750 --> 00:10:16.420 So you can't possibly kick energy out at a higher energy. 00:10:16.420 --> 00:10:24.700 And so this is a shorter wavelength in nanometers. 00:10:24.700 --> 00:10:26.926 And this is at a longer wavelength. 00:10:31.180 --> 00:10:34.640 So remember, higher energy are shorter wavelength. 00:10:34.640 --> 00:10:37.340 Lower energy are longer wavelengths. 00:10:37.340 --> 00:10:39.650 And that is a rule for fluorescence. 00:10:39.650 --> 00:10:41.510 When you excite a molecule, you'll 00:10:41.510 --> 00:10:43.430 take it to the excited state. 00:10:43.430 --> 00:10:45.680 It'll sit and vibrate there a little bit. 00:10:45.680 --> 00:10:48.590 Then it will kick back energy out at a longer wavelength. 00:10:48.590 --> 00:10:52.220 And for the majority of the fluorescence experiments 00:10:52.220 --> 00:10:55.070 that we do in biology, the wavelengths 00:10:55.070 --> 00:11:03.020 that you see emission at are in the visible range. 00:11:03.020 --> 00:11:07.280 Whereas the wavelengths that you might excite your molecule at 00:11:07.280 --> 00:11:16.740 are often in the UV or a bit longer, ideally, longer. 00:11:16.740 --> 00:11:18.597 And these things are going to come up. 00:11:18.597 --> 00:11:20.930 This isn't the first time that you're going to see them. 00:11:20.930 --> 00:11:23.710 And it's not the last time you're going to see them. 00:11:23.710 --> 00:11:26.270 So let's take a look at fluorescent dyes 00:11:26.270 --> 00:11:29.940 in the electromagnetic spectrum in the next couple of slides. 00:11:29.940 --> 00:11:39.580 So what you see here, you see a bunch of little eppendorf vials 00:11:39.580 --> 00:11:42.700 with fluorescent molecules that emit light 00:11:42.700 --> 00:11:44.680 at all different wavelengths. 00:11:44.680 --> 00:11:47.110 These would be down at the ultraviolet end 00:11:47.110 --> 00:11:49.480 of the electromagnetic spectrum. 00:11:49.480 --> 00:11:53.920 These would be up in the very red end, the lowest energy. 00:11:53.920 --> 00:11:56.560 So these emission wavelengths, these 00:11:56.560 --> 00:12:00.880 would be emitting at the lowest energy, longest wavelength. 00:12:00.880 --> 00:12:04.780 These would be emitting at the shortest wavelength, lowest 00:12:04.780 --> 00:12:06.268 to highest energy. 00:12:06.268 --> 00:12:07.310 Is everyone following me? 00:12:07.310 --> 00:12:09.180 So just make sure you remember that. 00:12:09.180 --> 00:12:12.490 And just this principle rule that you can't possibly 00:12:12.490 --> 00:12:15.160 break with respect to the wavelengths of light 00:12:15.160 --> 00:12:17.290 for fluorescence experiments. 00:12:17.290 --> 00:12:20.710 So what we're going to see is the relationship 00:12:20.710 --> 00:12:22.140 for the electromagnetic spectrum. 00:12:22.140 --> 00:12:24.130 There's a little bit more detail in a minute. 00:12:24.130 --> 00:12:26.110 And then look at some fluorescent dyes 00:12:26.110 --> 00:12:29.240 that are very, very commonly used in biology. 00:12:29.240 --> 00:12:31.690 And in fact, you will have seen a lot 00:12:31.690 --> 00:12:35.500 of cells stained with these dyes even so far in pictures 00:12:35.500 --> 00:12:37.300 that you've seen on the screen. 00:12:37.300 --> 00:12:41.020 And then we'll talk about the application of antibody 00:12:41.020 --> 00:12:43.300 reagents and where does that come in with respect 00:12:43.300 --> 00:12:44.590 to fluorescences. 00:12:44.590 --> 00:12:46.570 So let's take a look at fluorescence 00:12:46.570 --> 00:12:48.520 and the electromagnetic spectrum. 00:12:48.520 --> 00:12:52.720 So here it is, going from wavelengths. 00:12:52.720 --> 00:12:55.870 The ultraviolet wavelengths would be shorter 00:12:55.870 --> 00:12:58.420 than 400 nanometers. 00:12:58.420 --> 00:13:01.300 So ultraviolet, so beyond violet. 00:13:01.300 --> 00:13:05.230 And the very red wavelengths would be in the range 00:13:05.230 --> 00:13:07.330 from 600 to 700. 00:13:07.330 --> 00:13:09.580 And here you see the relationship 00:13:09.580 --> 00:13:15.640 between wavelength, and then what the light emitted 00:13:15.640 --> 00:13:16.610 would look like. 00:13:16.610 --> 00:13:20.230 So if we're looking at here, what we would expect to see 00:13:20.230 --> 00:13:23.930 is if we've got a fluorophore and it shows fluorescence, 00:13:23.930 --> 00:13:26.530 we would be exciting the fluorophore 00:13:26.530 --> 00:13:28.530 in wavelengths in this region. 00:13:28.530 --> 00:13:31.510 And we would see emission in wavelengths in this region. 00:13:31.510 --> 00:13:36.040 But a cardinal rule is that we excite with a shorter, higher 00:13:36.040 --> 00:13:38.950 energy wavelength and observe emission 00:13:38.950 --> 00:13:42.020 at a longer, lower energy wavelength. 00:13:42.020 --> 00:13:45.220 Now, there's very important dyes. 00:13:45.220 --> 00:13:47.500 Straight away the most common dye 00:13:47.500 --> 00:13:50.800 that you will see immediately in biology 00:13:50.800 --> 00:13:52.900 is a dye known as ethidium bromide. 00:13:52.900 --> 00:13:54.640 And here's its structure. 00:13:54.640 --> 00:13:57.190 You can often recognize fluorophores. 00:13:57.190 --> 00:14:00.640 They have lots of rings fused together with lots 00:14:00.640 --> 00:14:02.450 of double bonds in them. 00:14:02.450 --> 00:14:04.240 This is the structure of a compound 00:14:04.240 --> 00:14:06.610 known as ethidium bromide. 00:14:06.610 --> 00:14:11.480 It's a dye that intercalates into DNA. 00:14:11.480 --> 00:14:13.900 And when the dye changes its environment 00:14:13.900 --> 00:14:19.390 from being in water to being snuggled in between stacks 00:14:19.390 --> 00:14:23.920 of base pairs in DNA, it changes its fluorescent properties 00:14:23.920 --> 00:14:26.020 and it becomes fluorescence. 00:14:26.020 --> 00:14:30.290 So fluorescence isn't just the intrinsic shape of the molecule 00:14:30.290 --> 00:14:31.630 and what it looks like. 00:14:31.630 --> 00:14:35.980 It's very, very often related to what's around it. 00:14:35.980 --> 00:14:37.520 Why is that the case? 00:14:37.520 --> 00:14:41.680 It's because the excited state may behave differently 00:14:41.680 --> 00:14:43.420 in different environments. 00:14:43.420 --> 00:14:45.940 Maybe stabilize for a while, and that's 00:14:45.940 --> 00:14:49.330 why you might see fluorophores experience 00:14:49.330 --> 00:14:51.910 a change in their fluorescence as a function 00:14:51.910 --> 00:14:53.100 of their environment. 00:14:53.100 --> 00:14:54.580 Is that clear to everyone? 00:14:54.580 --> 00:14:57.550 So the molecular environments, if I'm a fluorophore 00:14:57.550 --> 00:15:00.220 and I'm in water, I'm going to feel pretty differently 00:15:00.220 --> 00:15:02.620 in my excited state if I'm a fluorophore 00:15:02.620 --> 00:15:06.100 and I'm sitting packed between DNA bases. 00:15:06.100 --> 00:15:08.230 It's pretty dramatic when you see it. 00:15:08.230 --> 00:15:11.590 So when you mix ethidium bromide with DNA, 00:15:11.590 --> 00:15:15.550 and it could be in a cell or it could be a lysate from a cell 00:15:15.550 --> 00:15:19.120 where you're capturing the DNA and trying to manipulate it 00:15:19.120 --> 00:15:21.130 in recombinant biology. 00:15:21.130 --> 00:15:25.180 That ethidium bromide will intercalate into the DNA. 00:15:25.180 --> 00:15:28.840 And it will light up as a bright orange dye. 00:15:28.840 --> 00:15:34.330 So here, say we've got a gel that we've run DNA on. 00:15:34.330 --> 00:15:36.850 We might have a set of standards. 00:15:36.850 --> 00:15:40.690 And in other places, we're looking for the size of DNA. 00:15:40.690 --> 00:15:42.430 Remember that great experiment we 00:15:42.430 --> 00:15:46.720 saw where we saw how quickly small and large pieces of DNA 00:15:46.720 --> 00:15:48.910 ran through an agarose gel? 00:15:48.910 --> 00:15:52.030 So here is what the DNA gel would 00:15:52.030 --> 00:15:56.110 look like if you soaked it ethidium bromide. 00:15:56.110 --> 00:15:58.600 So here's the gel as a ladder of bands. 00:15:58.600 --> 00:16:02.950 But then let's say you wanted to do some work on a piece of DNA 00:16:02.950 --> 00:16:07.960 and maybe ligated you see DNA pieces at different wavelengths 00:16:07.960 --> 00:16:11.080 that have different mobilities based on size. 00:16:11.080 --> 00:16:14.202 We couldn't see the DNA directly. 00:16:14.202 --> 00:16:15.160 We couldn't pick it up. 00:16:15.160 --> 00:16:16.600 We couldn't visualize it. 00:16:16.600 --> 00:16:19.190 So we have to use ways to visualize it. 00:16:19.190 --> 00:16:21.630 One way is to radio label it. 00:16:21.630 --> 00:16:23.830 Messy, we don't really want to do a lot of that 00:16:23.830 --> 00:16:25.060 if we can avoid it. 00:16:25.060 --> 00:16:28.450 The other way is simply to soak a dye into the gel. 00:16:28.450 --> 00:16:31.780 And the dye, because of the positive charge here, 00:16:31.780 --> 00:16:33.790 the counter ion gets displaced. 00:16:33.790 --> 00:16:36.130 And the positive charge gets attracted 00:16:36.130 --> 00:16:39.970 to the DNA and associates with it quite tightly. 00:16:39.970 --> 00:16:43.810 So that would be a way that you would observe 00:16:43.810 --> 00:16:47.200 DNA bound to dye in a gel. 00:16:47.200 --> 00:16:50.270 And this fluoresces a pretty long wavelength. 00:16:50.270 --> 00:16:52.690 So this fluoresces this bright orange 00:16:52.690 --> 00:16:54.970 that's actually at about 605. 00:16:54.970 --> 00:16:57.490 So you can see, this is really in the visible range. 00:16:57.490 --> 00:16:59.770 605 would be right around here. 00:16:59.770 --> 00:17:02.950 And it has this bright kind of orange fluorescence. 00:17:02.950 --> 00:17:04.329 And the wavelength that you would 00:17:04.329 --> 00:17:07.390 use to irradiate the dye on the DNA 00:17:07.390 --> 00:17:10.390 would be a shorter wavelength than 605. 00:17:10.390 --> 00:17:12.099 You will often have a prescription, 00:17:12.099 --> 00:17:15.970 excite at this wavelength so you observe at this wavelength. 00:17:15.970 --> 00:17:18.770 And these are fixed physical parameters 00:17:18.770 --> 00:17:21.550 for fluorescent molecules. 00:17:21.550 --> 00:17:27.790 Now, ethidium bromide is a dye that can get into cells. 00:17:27.790 --> 00:17:30.790 And we can look at DNA within cells. 00:17:30.790 --> 00:17:33.100 And here's a picture of how it would look. 00:17:33.100 --> 00:17:35.410 So here's the ethidium bromide. 00:17:35.410 --> 00:17:39.328 And here's a pair of stacked bases, this one and this one. 00:17:39.328 --> 00:17:40.870 And there he is, right in the middle. 00:17:40.870 --> 00:17:44.560 See that ring coming towards you is that. 00:17:44.560 --> 00:17:47.860 And then here's this thing that slides straight 00:17:47.860 --> 00:17:51.130 between the bases and might cause a little bit of a bulge. 00:17:51.130 --> 00:17:54.460 And we would call this a DNA intercalator. 00:17:54.460 --> 00:17:56.650 It slides into the DNA. 00:17:56.650 --> 00:17:59.170 And you can see over here, the structure of DNA. 00:17:59.170 --> 00:18:01.630 So you could picture ethidium bromide 00:18:01.630 --> 00:18:03.770 sliding between the bases. 00:18:03.770 --> 00:18:06.100 Now, there's a big problem here. 00:18:06.100 --> 00:18:10.630 Because you can't use DNA ethidium bromide. 00:18:10.630 --> 00:18:11.968 It's pretty toxic. 00:18:11.968 --> 00:18:12.885 Why would it be toxic? 00:18:18.210 --> 00:18:19.700 Well, no, it's not the bromide. 00:18:19.700 --> 00:18:22.880 It's the fact, more, think of what the dye does 00:18:22.880 --> 00:18:24.350 when it gets to the DNA. 00:18:24.350 --> 00:18:27.290 What would that do to things like replication 00:18:27.290 --> 00:18:28.850 and transcription? 00:18:28.850 --> 00:18:30.620 It just kind of messes it up. 00:18:30.620 --> 00:18:34.910 And so these are toxic dyes that can only be used in fixed cells 00:18:34.910 --> 00:18:37.500 to do observations of cells. 00:18:37.500 --> 00:18:39.110 So we use it a lot. 00:18:39.110 --> 00:18:41.600 We need to be careful of it because if it 00:18:41.600 --> 00:18:45.600 gets absorbed through our skin, it could get into our cells. 00:18:45.600 --> 00:18:47.630 And it could interfere with replication 00:18:47.630 --> 00:18:49.430 and other cellular processes. 00:18:49.430 --> 00:18:51.980 Because it would accumulate on our cellular DNA. 00:18:57.630 --> 00:19:00.350 And in fact, the interesting thing 00:19:00.350 --> 00:19:03.020 that a lot of molecules that actually 00:19:03.020 --> 00:19:05.630 have these sort of flat, pancake shapes 00:19:05.630 --> 00:19:08.000 with lots of double bonds are actually 00:19:08.000 --> 00:19:10.130 pretty important in biology. 00:19:10.130 --> 00:19:13.490 Because they end up being chemotherapeutic agents. 00:19:13.490 --> 00:19:15.890 So what I've shown you here is what's 00:19:15.890 --> 00:19:18.230 known as an anthracycline structure. 00:19:18.230 --> 00:19:20.870 I believe this is adriamycin, I could be off. 00:19:20.870 --> 00:19:26.760 But it's a natural product that's isolated from bacteria. 00:19:26.760 --> 00:19:29.210 And it has this structure that also 00:19:29.210 --> 00:19:31.880 makes it a DNA intercalator. 00:19:31.880 --> 00:19:35.240 And it's used as a cancer chemotherapeutic agent 00:19:35.240 --> 00:19:39.290 because it interferes with cell division and proliferation. 00:19:39.290 --> 00:19:42.140 So we actually exploit that property. 00:19:42.140 --> 00:19:46.670 But only with cells that we want to kill or stop dividing. 00:19:46.670 --> 00:19:48.320 So you could picture, well, I don't 00:19:48.320 --> 00:19:51.110 want to use something that's going 00:19:51.110 --> 00:19:53.810 to interfere with cells if I'm doing live cell imaging. 00:19:53.810 --> 00:19:56.120 Because I'm going to have trouble with the properties 00:19:56.120 --> 00:19:57.128 of the cells. 00:19:57.128 --> 00:19:58.670 So fluorophores are great, but you've 00:19:58.670 --> 00:20:03.200 got to worry about them because they can get transferred 00:20:03.200 --> 00:20:05.840 through the skin, through cellular membranes 00:20:05.840 --> 00:20:08.060 because they're often quite greasy. 00:20:08.060 --> 00:20:10.070 And they can get in and interfere 00:20:10.070 --> 00:20:12.650 with essential processes of DNA. 00:20:12.650 --> 00:20:16.220 So because of this, there's been quite a revolution in the work 00:20:16.220 --> 00:20:20.270 done with DNA binding agents that bind a little differently 00:20:20.270 --> 00:20:22.280 and are way less toxic. 00:20:22.280 --> 00:20:25.760 So I want to describe to you a series of dyes that are known 00:20:25.760 --> 00:20:32.350 as DAPI and HOECHST, H-O-E-C-H-S-T. This was, 00:20:32.350 --> 00:20:36.830 I believe, discovered in a bio company in Germany. 00:20:36.830 --> 00:20:40.670 And these are different kinds of dyes that 00:20:40.670 --> 00:20:43.130 fluoresce on binding to DNA. 00:20:43.130 --> 00:20:45.890 So they are still useful in that same context. 00:20:45.890 --> 00:20:49.100 You can in fact substitute ethidium bromide 00:20:49.100 --> 00:20:50.390 with these dyes. 00:20:50.390 --> 00:20:53.000 But they bind to DNA pretty differently. 00:20:53.000 --> 00:20:55.430 And I want you to take a look at these pictures. 00:20:55.430 --> 00:20:59.810 So here in green and blue-- and apologies to the color blind-- 00:20:59.810 --> 00:21:06.350 you see a molecule of this compound here bound to DNA. 00:21:06.350 --> 00:21:10.100 So it's pretty clear it's different from intercalation, 00:21:10.100 --> 00:21:11.060 right? 00:21:11.060 --> 00:21:16.080 You can see it's more sliding around one part of the DNA. 00:21:16.080 --> 00:21:19.370 And when those molecules bind to DNA in water, 00:21:19.370 --> 00:21:20.570 they don't fluorescence. 00:21:20.570 --> 00:21:24.410 When they bind to DNA they fluorescent an intense cyan 00:21:24.410 --> 00:21:25.700 blue. 00:21:25.700 --> 00:21:28.205 So that's at a shorter wavelength from the ethidium 00:21:28.205 --> 00:21:30.030 bromide. 00:21:30.030 --> 00:21:31.910 So taking a look at this structure, 00:21:31.910 --> 00:21:36.140 does anyone want to explain to me how the molecules might 00:21:36.140 --> 00:21:38.450 bind to DNA? 00:21:38.450 --> 00:21:41.270 We wouldn't call it intercalation. 00:21:41.270 --> 00:21:46.190 We know intercalation is perpendicular to the axis 00:21:46.190 --> 00:21:47.550 of the DNA. 00:21:47.550 --> 00:21:51.530 So where, looking at this, do you think these bind? 00:21:51.530 --> 00:21:54.710 A while ago when I was talking about the structure of DNA, 00:21:54.710 --> 00:22:00.050 I like to think of DNA as having two grooves, two places where 00:22:00.050 --> 00:22:01.280 things combine to it. 00:22:01.280 --> 00:22:03.740 And that's a minor groove. 00:22:03.740 --> 00:22:07.190 And then this big trench is what's called the major groove. 00:22:07.190 --> 00:22:09.740 And certain molecules bind in one groove. 00:22:09.740 --> 00:22:11.437 And other molecules bind in the other. 00:22:11.437 --> 00:22:12.770 Where do you think it's binding? 00:22:12.770 --> 00:22:13.850 Just by inspection. 00:22:16.540 --> 00:22:17.040 Yeah. 00:22:17.040 --> 00:22:18.110 AUDIENCE: Looks like it's in the minor groove. 00:22:18.110 --> 00:22:21.200 PROFESSOR: That's correct, it's just snuggled just perfectly 00:22:21.200 --> 00:22:22.105 in the minor groove. 00:22:22.105 --> 00:22:23.480 If it was in the major groove, it 00:22:23.480 --> 00:22:25.310 would be swimming around in that groove. 00:22:25.310 --> 00:22:27.290 It's almost too big. 00:22:27.290 --> 00:22:29.810 So what's really cool about these dyes 00:22:29.810 --> 00:22:34.640 is they slide in between into the minor groove. 00:22:34.640 --> 00:22:38.470 And they also make some contacts with the phosphodiester 00:22:38.470 --> 00:22:38.970 backbone. 00:22:38.970 --> 00:22:41.990 But it's not that they're dancing on the phosphodiester 00:22:41.990 --> 00:22:42.740 backbone. 00:22:42.740 --> 00:22:44.630 They're literally in the groove. 00:22:44.630 --> 00:22:48.290 But there's some opportunity for electrostatic interactions. 00:22:48.290 --> 00:22:51.380 And so these compounds would be known 00:22:51.380 --> 00:22:53.480 to bind in the minor groove. 00:22:53.480 --> 00:22:56.510 And in fact, they bind in particular regions of DNA 00:22:56.510 --> 00:22:59.030 where there's AT, not GC. 00:22:59.030 --> 00:23:01.020 Those are the places where there's just 00:23:01.020 --> 00:23:03.890 the pair of hydrogen bonds instead of the trio. 00:23:03.890 --> 00:23:07.310 That's just their habit, their personality. 00:23:07.310 --> 00:23:10.760 So chemistry was very important here. 00:23:10.760 --> 00:23:13.370 You had a good dye, but it was toxic. 00:23:13.370 --> 00:23:15.440 But improved dyes came along that could 00:23:15.440 --> 00:23:18.800 be used in living systems that are not toxic. 00:23:18.800 --> 00:23:20.690 Because if you bind in those grooves 00:23:20.690 --> 00:23:22.670 and you're dissociating easily, you're 00:23:22.670 --> 00:23:25.660 not going to interfere so much with replication. 00:23:25.660 --> 00:23:27.500 Does that make sense? 00:23:27.500 --> 00:23:29.120 So it's a weaker force. 00:23:29.120 --> 00:23:31.970 It's not going to have a big detrimental effect. 00:23:31.970 --> 00:23:33.620 Now, I moved this slide up. 00:23:33.620 --> 00:23:36.770 I realized he was in the wrong place in the deck. 00:23:36.770 --> 00:23:42.700 This is just an application of the DNA minor groove binder 00:23:42.700 --> 00:23:43.640 CEOCHST. 00:23:43.640 --> 00:23:47.450 And in this case, we're looking at three cells. 00:23:47.450 --> 00:23:51.350 These two are not actively dividing. 00:23:51.350 --> 00:23:53.540 But take a look at this cell, it's 00:23:53.540 --> 00:23:59.480 actually clearly in the state preparing for cell division. 00:23:59.480 --> 00:24:04.290 And what you can see here is that in the nucleus, 00:24:04.290 --> 00:24:07.610 the DNA is pretty diffuse before things really 00:24:07.610 --> 00:24:12.980 start to condense and line up for DNA replication and cell 00:24:12.980 --> 00:24:13.940 division. 00:24:13.940 --> 00:24:16.970 And what's intriguing to me is that this rather 00:24:16.970 --> 00:24:19.850 diffuse blue dye, that's probably a bit 00:24:19.850 --> 00:24:22.400 more loosely associated with DNA, 00:24:22.400 --> 00:24:25.520 becomes much clearer and brighter when 00:24:25.520 --> 00:24:29.070 the chromosomes are in the state they're in for cell division. 00:24:29.070 --> 00:24:30.940 So you can see them here. 00:24:30.940 --> 00:24:32.667 And one question here. 00:24:32.667 --> 00:24:34.250 So, if you're looking at cells, you're 00:24:34.250 --> 00:24:36.860 trying to observe cells, where else in the cell 00:24:36.860 --> 00:24:37.955 are you going to see DNA? 00:24:40.540 --> 00:24:42.730 So we can see the nuclear DNA, we 00:24:42.730 --> 00:24:45.100 can see what stage of the cell cycle it's in-- 00:24:45.100 --> 00:24:45.790 Yeah, Carmen. 00:24:45.790 --> 00:24:46.630 AUDIENCE: The mitochondria. 00:24:46.630 --> 00:24:48.172 PROFESSOR: Yeah, in the mitochondria. 00:24:48.172 --> 00:24:51.230 So you could also spot that within the cell 00:24:51.230 --> 00:24:53.860 if you're at a sufficient amplification. 00:24:53.860 --> 00:24:57.220 So you know that there's not DNA running around everywhere. 00:24:57.220 --> 00:25:01.330 It's literally in very specific places with the cell. 00:25:01.330 --> 00:25:06.010 These dyes will bind also to other nucleic acids. 00:25:06.010 --> 00:25:07.540 But they don't bind so well. 00:25:07.540 --> 00:25:10.660 Because those don't have the really repetitive, 00:25:10.660 --> 00:25:14.770 double stranded nucleotide structures. 00:25:14.770 --> 00:25:18.820 But there are other dyes that bind much more specifically 00:25:18.820 --> 00:25:19.600 to RNA. 00:25:19.600 --> 00:25:21.040 But we won't discuss them. 00:25:21.040 --> 00:25:22.930 So nucleic acids seem to be something 00:25:22.930 --> 00:25:25.970 that we can definitely pinpoint with fluorescence. 00:25:25.970 --> 00:25:27.310 We can see where it is. 00:25:27.310 --> 00:25:29.410 We could follow cell division. 00:25:29.410 --> 00:25:32.380 We could look to see the progress of cell division. 00:25:32.380 --> 00:25:36.430 For example, upon adding things to a cell, can you see-- 00:25:36.430 --> 00:25:39.580 remember very, very early on, we showed you 00:25:39.580 --> 00:25:41.410 movies of cells dividing. 00:25:41.410 --> 00:25:43.780 You could do that with this kind of dye 00:25:43.780 --> 00:25:46.960 because it's a non-toxic dye. 00:25:46.960 --> 00:25:49.780 So, great, so far, so good. 00:25:49.780 --> 00:25:52.870 So the key thing, though, about biology 00:25:52.870 --> 00:25:56.200 is we have so many other entities within a cell 00:25:56.200 --> 00:25:59.080 that we want to be able to track and monitor. 00:25:59.080 --> 00:26:02.500 And what we needed, what is absolutely essential 00:26:02.500 --> 00:26:05.720 are reagents to do that. 00:26:05.720 --> 00:26:18.900 So I want to talk to you about biological tools of monitoring. 00:26:21.978 --> 00:26:23.145 And you know what these are? 00:26:23.145 --> 00:26:26.520 These are antibodies, monitoring proteins. 00:26:26.520 --> 00:26:30.120 And in fact, you can also coax antibodies 00:26:30.120 --> 00:26:31.650 to recognize carbohydrates. 00:26:31.650 --> 00:26:33.330 So I'm going to just put these here. 00:26:40.500 --> 00:26:43.270 But they're a little bit harder to bind to antibodies. 00:26:43.270 --> 00:26:44.980 But nevertheless, those are useful. 00:26:44.980 --> 00:26:48.620 So we're going to talk now about antibodies, 00:26:48.620 --> 00:26:52.225 which are agents of the human adaptive immune system. 00:27:10.620 --> 00:27:16.350 And how they have been exploited intensively to study biology. 00:27:16.350 --> 00:27:20.880 Now, what you will learn from Professor Martin in two 00:27:20.880 --> 00:27:23.310 or three lectures time is much more 00:27:23.310 --> 00:27:26.340 about the nuts and bolts of the immune cells, 00:27:26.340 --> 00:27:27.870 of the immune system. 00:27:27.870 --> 00:27:32.010 And how it mounts a response to disease and other features. 00:27:32.010 --> 00:27:35.160 I'm going to focus completely on the technological side 00:27:35.160 --> 00:27:37.470 of antibodies and how they are useful 00:27:37.470 --> 00:27:39.990 reagents to study biology. 00:27:39.990 --> 00:27:44.460 Because if you want to recognize a protein in a cell, 00:27:44.460 --> 00:27:48.270 you need a particular entity that will bind to that protein 00:27:48.270 --> 00:27:50.850 and show you where it is through some kind of signal, 00:27:50.850 --> 00:27:52.680 for example, fluorescence. 00:27:52.680 --> 00:27:55.650 So what I want to do is give you the minimal description 00:27:55.650 --> 00:27:58.230 of antibodies so you can understand this. 00:27:58.230 --> 00:28:00.600 But later you're going to revisit it 00:28:00.600 --> 00:28:02.730 in a bit more complicated venue. 00:28:02.730 --> 00:28:04.830 But for the time being, I'm just going 00:28:04.830 --> 00:28:12.970 to talk about B cells, which are cells that produce antibodies. 00:28:17.090 --> 00:28:19.580 And I'm going to talk to you about how 00:28:19.580 --> 00:28:22.730 they recognize their targets. 00:28:22.730 --> 00:28:25.850 Because what we have in the adaptive immune system 00:28:25.850 --> 00:28:30.530 is an amazing system where you can do combinatorial biology 00:28:30.530 --> 00:28:33.420 and basically recognize any target entity 00:28:33.420 --> 00:28:34.730 you're interested in. 00:28:34.730 --> 00:28:37.790 So let's take a look at this, keeping in mind 00:28:37.790 --> 00:28:40.490 that this is us exploiting biology 00:28:40.490 --> 00:28:42.920 to make reagents to do biology. 00:28:42.920 --> 00:28:45.740 It's kind of a cool sort of cyclical process. 00:28:45.740 --> 00:28:49.940 So the cells of the hematopoietic immune-- 00:28:49.940 --> 00:28:52.910 sorry the hematopoietic system, those are the ones that are 00:28:52.910 --> 00:28:57.680 important in blood cells, form a lot of different-- 00:28:57.680 --> 00:28:59.750 wait a minute, I'm on triple-- 00:28:59.750 --> 00:29:00.960 triple tools here. 00:29:00.960 --> 00:29:02.660 There are a bunch of different cells 00:29:02.660 --> 00:29:05.840 that are produced from the pluripotent hematopoietic 00:29:05.840 --> 00:29:06.480 cells. 00:29:06.480 --> 00:29:09.410 They're either the white cells or the red cell types. 00:29:09.410 --> 00:29:13.470 But what we're going to focus right in on are the B cells. 00:29:13.470 --> 00:29:15.530 These are the cells of the immune system that 00:29:15.530 --> 00:29:18.350 produce soluble antibodies, OK. 00:29:21.260 --> 00:29:25.160 And when you challenge a B cell population 00:29:25.160 --> 00:29:29.330 with a foreign entity, the B cell population 00:29:29.330 --> 00:29:32.560 will go into gear to produce antibodies 00:29:32.560 --> 00:29:37.220 that very specifically recognize that foreign target, because 00:29:37.220 --> 00:29:39.800 in the human adaptive immune system, that 00:29:39.800 --> 00:29:44.190 might be a wonderful tool to get rid of that foreign entity. 00:29:44.190 --> 00:29:45.830 So we're going to focus exclusively 00:29:45.830 --> 00:29:49.430 on the B cells and the way that they mature 00:29:49.430 --> 00:29:52.760 to produce soluble antibodies, those are down at the bottom 00:29:52.760 --> 00:29:56.120 here, based on what they've been challenged with. 00:29:56.120 --> 00:29:58.880 So what this little schematic shows you is you 00:29:58.880 --> 00:30:02.780 have a bunch of different B cells. 00:30:02.780 --> 00:30:05.150 And there's something you want to recognize. 00:30:05.150 --> 00:30:08.770 Let's just say it's a molecule-- 00:30:08.770 --> 00:30:11.540 an EGF molecule, a cytokine. 00:30:11.540 --> 00:30:15.170 What you might do is challenge this population 00:30:15.170 --> 00:30:16.700 with the cytokine. 00:30:16.700 --> 00:30:20.630 Only one B cell type will bind to the cytokine. 00:30:20.630 --> 00:30:23.130 And then that will get amplified. 00:30:23.130 --> 00:30:25.700 And then you will end up with B cells 00:30:25.700 --> 00:30:28.010 that produce a lot of an antibody 00:30:28.010 --> 00:30:31.610 to a cytokine such as EGF. 00:30:31.610 --> 00:30:34.280 I'm truly dumbing this down. 00:30:34.280 --> 00:30:36.140 I just want you to get the gist of it 00:30:36.140 --> 00:30:38.750 for the purpose of this discussion. 00:30:38.750 --> 00:30:42.940 Now B cells adopt a very classical shape. 00:30:42.940 --> 00:30:47.270 And I'm just going to show you the quaternary structure of a B 00:30:47.270 --> 00:30:47.810 cell-- 00:30:47.810 --> 00:30:54.140 of an antibody in linear form here. 00:30:54.140 --> 00:30:56.630 So it doesn't look very exciting right now. 00:30:56.630 --> 00:31:06.040 There are two light chains, which I've just 00:31:06.040 --> 00:31:08.410 shown in schematic form. 00:31:08.410 --> 00:31:11.440 These are just polypeptide chains. 00:31:11.440 --> 00:31:13.060 And there are two heavy chains. 00:31:19.720 --> 00:31:22.820 All right, so that's their basic structure. 00:31:22.820 --> 00:31:25.940 It's held together in a stable quarternary 00:31:25.940 --> 00:31:28.940 structure with this complex. 00:31:28.940 --> 00:31:33.200 And there may be disulfides across and throughout 00:31:33.200 --> 00:31:34.280 the structure. 00:31:34.280 --> 00:31:36.530 Now what's so special about antibodies? 00:31:36.530 --> 00:31:38.360 They're pretty big molecules. 00:31:38.360 --> 00:31:40.100 The molecular weight is pretty high. 00:31:44.520 --> 00:31:46.700 But the key thing about antibodies 00:31:46.700 --> 00:31:53.910 is that the majority of the structure 00:31:53.910 --> 00:31:55.455 stays fairly constant. 00:31:58.080 --> 00:31:59.300 So I'm just going to-- 00:31:59.300 --> 00:32:05.760 so this doesn't get modified when B cells mature. 00:32:05.760 --> 00:32:10.640 But another part of the structure is variable. 00:32:14.680 --> 00:32:17.680 And when B cells mature, there's loads 00:32:17.680 --> 00:32:20.860 of rearranging in that variable section in order 00:32:20.860 --> 00:32:24.050 that it adapt to bind to target. 00:32:24.050 --> 00:32:26.890 So what you've seen here is that the target-- 00:32:26.890 --> 00:32:28.660 see if this little fellow works anymore. 00:32:28.660 --> 00:32:29.160 No. 00:32:29.160 --> 00:32:30.730 Ah. 00:32:30.730 --> 00:32:32.890 What you see here is the light chain 00:32:32.890 --> 00:32:35.990 in green, heavy chain in blue. 00:32:35.990 --> 00:32:37.460 And it's a double version of it. 00:32:37.460 --> 00:32:39.910 We always draw antibodies as this V shape. 00:32:39.910 --> 00:32:49.890 And an antigen-- you've heard this word before-- 00:32:49.890 --> 00:32:53.625 it's a foreign entity that's foreign to the immune system. 00:33:02.490 --> 00:33:08.390 The antigen binding site is right here 00:33:08.390 --> 00:33:11.890 at the tips of the antibody. 00:33:11.890 --> 00:33:15.200 And I think the picture up there is kind of clearer. 00:33:15.200 --> 00:33:16.540 Let's put that forward again. 00:33:16.540 --> 00:33:19.630 And you can see very specifically the structure. 00:33:19.630 --> 00:33:22.120 The C's designate constant regions. 00:33:27.930 --> 00:33:30.360 See C all the way through here. 00:33:30.360 --> 00:33:34.290 And V's represent variable regions, which I've shown you. 00:33:34.290 --> 00:33:37.620 And at the tip of the V's are the antigen binding sites. 00:33:37.620 --> 00:33:39.780 So you're going to see more about antibodies 00:33:39.780 --> 00:33:41.252 in the immune system. 00:33:41.252 --> 00:33:42.960 But what you want to accept is that these 00:33:42.960 --> 00:33:47.040 are biological macromolecules that particularly evolved 00:33:47.040 --> 00:33:49.710 to recognize target antigens. 00:33:49.710 --> 00:33:55.660 And you can use them reliably as biological reagents. 00:33:55.660 --> 00:33:59.110 All right, so how do you achieve the diversity? 00:33:59.110 --> 00:34:02.290 There are hundreds of thousands of different antibodies 00:34:02.290 --> 00:34:03.850 in the human system. 00:34:03.850 --> 00:34:07.540 If we had a gene for every single different light 00:34:07.540 --> 00:34:11.080 chain and every heavy chain, you know, our DNA 00:34:11.080 --> 00:34:13.090 would be completely swamped by being 00:34:13.090 --> 00:34:17.679 dedicated to the genetic material for antibodies. 00:34:17.679 --> 00:34:20.949 So instead there is a particular system 00:34:20.949 --> 00:34:25.840 which provides little portions of the DNA structure 00:34:25.840 --> 00:34:29.210 that are in little pieces of variable components 00:34:29.210 --> 00:34:33.440 that can get zipped together through transcription 00:34:33.440 --> 00:34:37.810 and slicing events to give you a bunch of antibodies that have 00:34:37.810 --> 00:34:40.389 different variable regions. 00:34:40.389 --> 00:34:42.850 And this is what's known as the BDJ system. 00:34:42.850 --> 00:34:46.120 And you'll hear more about that from Professor Martin. 00:34:46.120 --> 00:34:48.070 So basically what you want to think about 00:34:48.070 --> 00:34:52.659 is it's a combinatorial system to take little pieces of DNA 00:34:52.659 --> 00:34:55.510 into a super molecular structure to give you 00:34:55.510 --> 00:34:59.320 antibody combining sites that can recognize virtually 00:34:59.320 --> 00:35:00.880 any target. 00:35:00.880 --> 00:35:02.480 Anyone got any questions here? 00:35:02.480 --> 00:35:06.630 I'm seeing a few worried faces. 00:35:06.630 --> 00:35:08.550 Somebody ask me a question if you 00:35:08.550 --> 00:35:10.830 feel I could clarify a component of this 00:35:10.830 --> 00:35:11.970 or are you OK with this? 00:35:15.610 --> 00:35:18.150 Anybody? 00:35:18.150 --> 00:35:20.060 OK, I'll move forward. 00:35:23.050 --> 00:35:25.600 So we talked about antibodies. 00:35:25.600 --> 00:35:27.670 When you get a population of B cells 00:35:27.670 --> 00:35:33.550 that produce antibodies to a particular target, 00:35:33.550 --> 00:35:36.070 these may be what are known as polyclonal-- 00:35:41.283 --> 00:35:43.700 a polyclonal-- sorry, I should have written that up here-- 00:35:43.700 --> 00:35:52.970 a polyclonal antibody, as might be suggested by the name, 00:35:52.970 --> 00:35:54.350 is an antibody-- 00:35:54.350 --> 00:35:58.460 let's say it recognizes a molecular entity. 00:35:58.460 --> 00:36:00.010 So the antibodies-- 00:36:00.010 --> 00:36:03.520 I'm going to draw them as little y shaped molecules-- 00:36:03.520 --> 00:36:09.970 may recognize different parts of the antigen. So antibody A, 00:36:09.970 --> 00:36:13.660 B, C recognize different parts of the antigen. 00:36:13.660 --> 00:36:15.280 Those would be polyclonal. 00:36:15.280 --> 00:36:19.750 It would be a mixed bag of antibody molecules 00:36:19.750 --> 00:36:22.750 that shows specificity for a target. 00:36:22.750 --> 00:36:27.156 But people also tend to use a great deal 00:36:27.156 --> 00:36:34.700 of monoclonal antibodies, because they 00:36:34.700 --> 00:36:36.500 are a lot more specific. 00:36:36.500 --> 00:36:40.780 So if you had a selection of polyclonal antibodies, 00:36:40.780 --> 00:36:44.330 a monoclonal antibody would be a single population 00:36:44.330 --> 00:36:47.600 that recognizes a single antigen or epitope 00:36:47.600 --> 00:36:49.520 in your antigenic molecule. 00:36:49.520 --> 00:36:53.510 And the way those are made is through the engineering method 00:36:53.510 --> 00:36:57.170 where you fuse spleen cells with myeloma cells. 00:36:57.170 --> 00:36:59.360 And then you get a hybrid-- 00:36:59.360 --> 00:37:03.050 what are known as hybridomas that produce very specifically 00:37:03.050 --> 00:37:05.210 just monoclonal antibodies. 00:37:05.210 --> 00:37:07.850 So that's a bit of background there about the antibodies. 00:37:07.850 --> 00:37:11.240 You'll see it on the rerun when Professor Martin talks. 00:37:11.240 --> 00:37:14.150 But I just wanted to give you a bit of exposure to this. 00:37:14.150 --> 00:37:18.360 So let's now look at how antibodies can be useful. 00:37:18.360 --> 00:37:21.290 So let's say you want to visualize in a cell-- let's 00:37:21.290 --> 00:37:24.050 move straight to a real targeted application. 00:37:24.050 --> 00:37:27.680 We want to make an antibody that might recognize actin 00:37:27.680 --> 00:37:31.100 and a different antibody that might recognize tubulin 00:37:31.100 --> 00:37:35.930 to take a look at cells through the use of antibody structures. 00:37:35.930 --> 00:37:39.860 The way you generate antibodies is through laboratory animals. 00:37:39.860 --> 00:37:43.700 And very commonly we use either mice or rabbits 00:37:43.700 --> 00:37:45.290 for antibody production. 00:37:47.810 --> 00:37:51.830 The rabbit is used when you need a lot more antibody material. 00:37:51.830 --> 00:37:55.020 The mouse will satisfy for some experiments. 00:37:55.020 --> 00:37:59.970 So the way you make antibodies is by injecting. 00:37:59.970 --> 00:38:03.530 So this would be the foreign agent or antigen 00:38:03.530 --> 00:38:05.570 that you want to make an antibody to, 00:38:05.570 --> 00:38:09.020 which would normally bind at the variable region 00:38:09.020 --> 00:38:11.390 of this immunoglobulin molecule. 00:38:11.390 --> 00:38:14.510 You inject the mouse with that antigen. Excuse 00:38:14.510 --> 00:38:15.533 me-- that's a rabbit. 00:38:15.533 --> 00:38:17.450 You know, I haven't been in biology very long. 00:38:17.450 --> 00:38:20.430 But I can tell that's a rabbit, right? 00:38:20.430 --> 00:38:26.185 Anyway, so you inject the rabbit with a human protein. 00:38:26.185 --> 00:38:27.560 If you-- what would happen if you 00:38:27.560 --> 00:38:35.450 injected the rabbit with rabbit actin or with rabbit tubulin? 00:38:35.450 --> 00:38:40.600 What would you-- would you expect to see a response? 00:38:40.600 --> 00:38:41.430 No, right? 00:38:41.430 --> 00:38:42.150 Yeah. 00:38:42.150 --> 00:38:45.840 Because the organisms are adapted 00:38:45.840 --> 00:38:48.510 not to recognize their own proteins 00:38:48.510 --> 00:38:51.850 unless there's some disorder like an autoimmune disease. 00:38:51.850 --> 00:38:56.470 So you would inject the rabbit with a human epitope, 00:38:56.470 --> 00:39:00.300 so for example human actin, generate antibodies 00:39:00.300 --> 00:39:03.150 with a specificity for actin, and then 00:39:03.150 --> 00:39:06.930 you would label those antibodies with a fluorescent marker, 00:39:06.930 --> 00:39:10.290 so you could track it or follow it through chemistry. 00:39:10.290 --> 00:39:13.620 Alternatively, you might want to make a different antibody 00:39:13.620 --> 00:39:14.970 for tubulin. 00:39:14.970 --> 00:39:18.750 And then you would differentiate the antibody against tubulin 00:39:18.750 --> 00:39:21.720 from the antibody against actin by labeling it 00:39:21.720 --> 00:39:24.000 with a different color fluorophore. 00:39:24.000 --> 00:39:27.390 So you've really got two types of macromolecule 00:39:27.390 --> 00:39:30.210 that can be interacted with a fixed cell 00:39:30.210 --> 00:39:32.490 and recognize those two macromolecules 00:39:32.490 --> 00:39:33.570 within the protein. 00:39:33.570 --> 00:39:35.670 So what's important here is that you 00:39:35.670 --> 00:39:38.400 don't use the protein from-- 00:39:38.400 --> 00:39:40.590 if you want to study human cells, 00:39:40.590 --> 00:39:43.800 you use antibodies produced in rabbit or mouse. 00:39:43.800 --> 00:39:45.900 If you want to study rabbit cells, 00:39:45.900 --> 00:39:48.500 you could produce it in different organisms. 00:39:48.500 --> 00:39:52.250 You're not going to produce the antibodies in the same host. 00:39:52.250 --> 00:39:53.900 Nowadays-- yes. 00:39:53.900 --> 00:39:56.854 AUDIENCE: How do you know that you have the correct antibody, 00:39:56.854 --> 00:40:00.600 like if that is the right one? 00:40:00.600 --> 00:40:01.770 PROFESSOR: Right. 00:40:01.770 --> 00:40:05.220 OK, so you would prepare the-- 00:40:05.220 --> 00:40:07.050 you would do them. 00:40:07.050 --> 00:40:09.150 There's a lot of screening takes place. 00:40:09.150 --> 00:40:11.670 So you inject the mouse or rabbit. 00:40:11.670 --> 00:40:13.260 And then you collect the serum. 00:40:13.260 --> 00:40:15.510 And you look for whether the serum 00:40:15.510 --> 00:40:17.820 gets enriched and enriched in antibodies 00:40:17.820 --> 00:40:20.040 that recognize the target. 00:40:20.040 --> 00:40:23.280 So you'll see an increase in what's known as the titer. 00:40:23.280 --> 00:40:26.040 And then once you get a high titer, you'll-- 00:40:26.040 --> 00:40:28.950 unfortunately, you can just collect the serum 00:40:28.950 --> 00:40:31.440 or depending if you want to make monoclonal, 00:40:31.440 --> 00:40:34.380 you'd have to sacrifice the animal to get the spleen cells. 00:40:34.380 --> 00:40:36.660 But then you collect the antibodies 00:40:36.660 --> 00:40:38.340 through an affinity method that's 00:40:38.340 --> 00:40:40.380 directed just at antibodies. 00:40:40.380 --> 00:40:41.790 Then you've got your population. 00:40:41.790 --> 00:40:44.400 And you can throw a fluorophore dye at it 00:40:44.400 --> 00:40:45.600 and chemically label it. 00:40:45.600 --> 00:40:47.280 So it's a good point there. 00:40:47.280 --> 00:40:48.840 There's a lot of work being done now 00:40:48.840 --> 00:40:51.660 with antibodies from different organisms, in fact, 00:40:51.660 --> 00:40:53.700 you'll see them from camels. 00:40:53.700 --> 00:40:55.743 And they're also ones from shark. 00:40:55.743 --> 00:40:57.660 And the reason why they're kind of interesting 00:40:57.660 --> 00:41:00.630 is that they sort of have mini antibodies that are much more 00:41:00.630 --> 00:41:02.590 useful for technology. 00:41:02.590 --> 00:41:05.020 So let's see what we can do here. 00:41:05.020 --> 00:41:08.100 We can do fluorescence experiments. 00:41:08.100 --> 00:41:10.450 In order to do this with an antibody. 00:41:10.450 --> 00:41:13.890 And this is going to highlight a shortcoming of antibodies. 00:41:13.890 --> 00:41:15.420 You may take a cell that you want 00:41:15.420 --> 00:41:18.310 to observe different proteins in that cell, 00:41:18.310 --> 00:41:21.750 you have to fix the cell to make it permeable. 00:41:21.750 --> 00:41:25.200 Why do I need to make the cell permeable 00:41:25.200 --> 00:41:27.450 in order to use these antibody reagents? 00:41:33.330 --> 00:41:36.770 Do you think the antibodies are just going to cross-- 00:41:36.770 --> 00:41:38.820 DAPI gets into a cell easily. 00:41:38.820 --> 00:41:41.860 But what about antibodies? 00:41:41.860 --> 00:41:43.570 Can they cross the plasma membrane 00:41:43.570 --> 00:41:45.340 to get into the cell to label a target? 00:41:48.530 --> 00:41:49.970 What do you think? 00:41:49.970 --> 00:41:53.020 Who says yes? 00:41:53.020 --> 00:41:54.140 Who says no? 00:41:54.140 --> 00:41:54.750 Good. 00:41:54.750 --> 00:41:55.580 Thank goodness. 00:41:55.580 --> 00:41:56.110 OK. 00:41:56.110 --> 00:41:57.110 You guys don't say much. 00:41:57.110 --> 00:41:58.610 But I know you know the answer here. 00:41:58.610 --> 00:42:00.260 They just can't float into cells. 00:42:00.260 --> 00:42:02.430 They're too large to get into cells. 00:42:02.430 --> 00:42:04.940 So you have to fix the cells on a glass side 00:42:04.940 --> 00:42:09.940 and permeabilize them, for example, with methanol 00:42:09.940 --> 00:42:12.230 so that the antibodies can gain access 00:42:12.230 --> 00:42:14.010 to all parts of the cell. 00:42:14.010 --> 00:42:17.570 So here is a bright field view of cells. 00:42:17.570 --> 00:42:20.270 That's a little bit more specific. 00:42:20.270 --> 00:42:21.740 That's OK too. 00:42:21.740 --> 00:42:23.780 But this is what I really want to show you. 00:42:23.780 --> 00:42:28.280 This is what you could achieve with DAPI and an antibody 00:42:28.280 --> 00:42:31.190 to the actin and an antibody to tubulin. 00:42:31.190 --> 00:42:33.290 And you can look at the various colors 00:42:33.290 --> 00:42:35.000 of the fluorescence emission. 00:42:35.000 --> 00:42:39.650 And you see here what you've got is an anti-actin antibody 00:42:39.650 --> 00:42:40.580 with a red dye. 00:42:40.580 --> 00:42:43.850 And you can see that at the perimeters of the cells. 00:42:43.850 --> 00:42:47.420 You've got an anti-tubulin antibody with a green dye. 00:42:47.420 --> 00:42:51.980 And you can see the filamentous structure there. 00:42:51.980 --> 00:42:55.040 And then you've got DAPI staining bright blue 00:42:55.040 --> 00:42:56.290 where the nuclei are. 00:42:56.290 --> 00:42:58.910 So you have three unique labels that 00:42:58.910 --> 00:43:00.590 fluoresce at different wavelengths 00:43:00.590 --> 00:43:02.810 and you can directly pinpoint things. 00:43:02.810 --> 00:43:06.430 So fluorescence is extremely valuable for looking 00:43:06.430 --> 00:43:09.760 a biological systems, because we don't have a lot that 00:43:09.760 --> 00:43:11.120 fluoresces in the body. 00:43:11.120 --> 00:43:14.360 So a cell in general, if you irradiate it with light, 00:43:14.360 --> 00:43:17.240 you won't see any major fluorescence at all. 00:43:17.240 --> 00:43:21.050 So these reagents that you use to study biology, 00:43:21.050 --> 00:43:24.470 if they fluoresce, you've got unique signals where you can 00:43:24.470 --> 00:43:28.040 look at really complicated cells that may have thousands 00:43:28.040 --> 00:43:31.010 and thousands of proteins, sugars, nucleic acids, 00:43:31.010 --> 00:43:34.760 and very specifically see things by fluorescence 00:43:34.760 --> 00:43:38.510 because the fluorescence is a unique signal in biology 00:43:38.510 --> 00:43:41.960 relative to the intrinsic fluorescence of proteins 00:43:41.960 --> 00:43:43.730 or rather macromolecules. 00:43:43.730 --> 00:43:47.390 We have tryptophan and tyrosine, a couple of amino acids. 00:43:47.390 --> 00:43:49.670 They fluoresce, but it's so dim. 00:43:49.670 --> 00:43:50.870 It's nothing like this. 00:43:50.870 --> 00:43:53.780 You wouldn't see those kinds of signals at all. 00:43:53.780 --> 00:43:57.410 It's really what we call extrinsic fluorophores, 00:43:57.410 --> 00:44:01.190 fluorophores from outside that shine very, very brightly. 00:44:01.190 --> 00:44:03.770 Many of these fluorophores shine so brightly they 00:44:03.770 --> 00:44:07.280 can be used to look at in single molecules-- 00:44:07.280 --> 00:44:08.930 professor Martin described to you 00:44:08.930 --> 00:44:11.750 single molecule DNA sequencing. 00:44:11.750 --> 00:44:15.080 That actually exploits very bright fluorophores 00:44:15.080 --> 00:44:17.450 that is so bright that you can see just a few of them 00:44:17.450 --> 00:44:20.900 in one place very, very clearly. 00:44:20.900 --> 00:44:22.700 OK, how am I doing? 00:44:22.700 --> 00:44:24.800 I just want to actually leave you 00:44:24.800 --> 00:44:27.410 with something that's another technology. 00:44:27.410 --> 00:44:29.420 So you could almost picture-- 00:44:29.420 --> 00:44:33.460 with all of what we've seen so far, 00:44:33.460 --> 00:44:39.560 you could almost target any cell with an antibody that's 00:44:39.560 --> 00:44:42.170 specifically raised to a particular protein that's 00:44:42.170 --> 00:44:43.400 within the cell. 00:44:43.400 --> 00:44:45.260 So we can see-- 00:44:45.260 --> 00:44:48.210 in the next class we'll discuss what the limitations of that 00:44:48.210 --> 00:44:48.710 are. 00:44:48.710 --> 00:44:50.780 But we've already talked about the fact 00:44:50.780 --> 00:44:54.940 that we have to use antibodies with fixed, not living anymore, 00:44:54.940 --> 00:44:55.760 cells. 00:44:55.760 --> 00:44:59.040 So they are really dyes that can only be used in that way. 00:44:59.040 --> 00:45:01.220 The other place you can use fluorophores 00:45:01.220 --> 00:45:04.910 is for labeling DNA and from looking at DNA. 00:45:04.910 --> 00:45:07.880 And a particularly important technology 00:45:07.880 --> 00:45:12.410 is known as a DNA microarray has anybody heard of these? 00:45:12.410 --> 00:45:12.920 Yeah. 00:45:12.920 --> 00:45:17.690 So DNA microarrays absolutely exploits the complementarity 00:45:17.690 --> 00:45:19.080 of DNA sequences. 00:45:19.080 --> 00:45:22.730 So if you want to probe for a particular sequence of DNA, 00:45:22.730 --> 00:45:25.490 you would make the complementary strand 00:45:25.490 --> 00:45:27.350 and label it with a fluorophore. 00:45:27.350 --> 00:45:31.730 And new could, out of thousands of DNA stretches, literally 00:45:31.730 --> 00:45:33.830 light up the stretch of DNA that's 00:45:33.830 --> 00:45:36.080 complementary to your target. 00:45:36.080 --> 00:45:38.270 And so DNA microarrays-- 00:45:38.270 --> 00:45:40.040 what you see here are just the size 00:45:40.040 --> 00:45:42.350 of just a microscope slide. 00:45:42.350 --> 00:45:47.690 On this slide through arranged technologies, 00:45:47.690 --> 00:45:53.570 you can literally spot 40,000 distinct sequences of DNA 00:45:53.570 --> 00:45:55.580 in grids to recognize. 00:45:55.580 --> 00:45:58.400 So these DNA microarrays can be used 00:45:58.400 --> 00:46:04.040 for profiling genetic material or for profiling not just DNA, 00:46:04.040 --> 00:46:06.590 but RNA, and we'll see how at the beginning 00:46:06.590 --> 00:46:10.700 of the next class, in order to probe for particular stretches 00:46:10.700 --> 00:46:14.240 of DNA that might be disease related 00:46:14.240 --> 00:46:17.810 and have single nucleotide polymorphisms. 00:46:17.810 --> 00:46:20.870 So in order to actually just give you a little bit of a warm 00:46:20.870 --> 00:46:23.180 up to that, I want you to go-- 00:46:23.180 --> 00:46:25.640 I'm going to put a link to this in the web site. 00:46:25.640 --> 00:46:30.380 And it actually just shows you a virtual running of a DNA 00:46:30.380 --> 00:46:32.300 microarray experiments and how you 00:46:32.300 --> 00:46:35.090 can use it to profile disease states 00:46:35.090 --> 00:46:37.760 and cells versus healthy cells. 00:46:37.760 --> 00:46:39.930 And then at the beginning of the next class, 00:46:39.930 --> 00:46:42.680 I'll describe how you get information out 00:46:42.680 --> 00:46:44.270 of DNA microarrays. 00:46:44.270 --> 00:46:46.520 But at the end of the day, you're 00:46:46.520 --> 00:46:49.700 always using the fluorophores as the probes 00:46:49.700 --> 00:46:52.090 for where certain things are, OK. 00:46:52.090 --> 00:46:53.860 And the thing to remember here. 00:46:53.860 --> 00:46:56.290 Fluorescence is a magnificent tool. 00:46:56.290 --> 00:46:58.610 We can use fluorophores on their own. 00:46:58.610 --> 00:47:01.390 We can use fluorophores as attached to antibodies. 00:47:01.390 --> 00:47:03.400 And the DNA microarray experiments 00:47:03.400 --> 00:47:05.590 show you how you can use fluorophores 00:47:05.590 --> 00:47:08.340 attached to DNA sequences, OK. 00:47:08.340 --> 00:47:10.880 And I'll see you in the next class.