WEBVTT 00:00:00.960 --> 00:00:03.270 The following content is provided under a Creative 00:00:03.270 --> 00:00:04.630 Commons license. 00:00:04.630 --> 00:00:07.140 Your support will help MIT OpenCourseWare 00:00:07.140 --> 00:00:11.470 continue to offer high-quality educational resources for free. 00:00:11.470 --> 00:00:14.100 To make a donation, or view additional materials 00:00:14.100 --> 00:00:18.050 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:18.050 --> 00:00:19.000 at ocw.mit.edu. 00:00:24.807 --> 00:00:26.640 JOANNE STUBBE: This is the second recitation 00:00:26.640 --> 00:00:29.250 on cholesterol, and it's really focused 00:00:29.250 --> 00:00:34.680 on this question of how do you sense cholesterol 00:00:34.680 --> 00:00:36.030 in a membrane? 00:00:36.030 --> 00:00:39.540 So that's really a tough problem. 00:00:39.540 --> 00:00:43.020 And they've developed new tools, and that's 00:00:43.020 --> 00:00:45.470 what we're going to be talking about-- what the tools are, 00:00:45.470 --> 00:00:48.390 and whether you would think they were 00:00:48.390 --> 00:00:53.400 adequate to be able to address this question about what kinds 00:00:53.400 --> 00:00:56.380 of changes in concentration of cholesterol. 00:00:56.380 --> 00:00:58.000 Number one, can you measure them? 00:00:58.000 --> 00:01:01.620 And number two, what effects do they have, 00:01:01.620 --> 00:01:05.220 in terms of whether you're going to turn on cholesterol 00:01:05.220 --> 00:01:08.760 biosynthesis and uptake, because you need more cholesterol, 00:01:08.760 --> 00:01:11.490 or you're going to turn the whole thing off? 00:01:11.490 --> 00:01:16.110 So we've been focusing, as we've described 00:01:16.110 --> 00:01:20.010 in the last few lectures, in the endoplasmic reticulum. 00:01:20.010 --> 00:01:23.400 And what would the cholesterol-- 00:01:23.400 --> 00:01:25.920 what kinds of changes in cholesterols 00:01:25.920 --> 00:01:28.890 did they see in the experiments they were doing in this paper? 00:01:28.890 --> 00:01:34.709 What were the range of changes that they saw? 00:01:34.709 --> 00:01:36.010 AUDIENCE: 3% to 10%? 00:01:36.010 --> 00:01:39.300 JOANNE STUBBE: Yeah, so see, something low. 00:01:39.300 --> 00:01:42.840 Say they were trying to do this same experiment in the plasma 00:01:42.840 --> 00:01:45.450 membrane-- how do we know it's the ER membrane that 00:01:45.450 --> 00:01:46.775 does this sensing? 00:01:46.775 --> 00:01:48.850 That's what the whole paper is focused on, 00:01:48.850 --> 00:01:51.960 that's what everything we've focused on in class. 00:01:51.960 --> 00:01:54.870 Say you wanted to do a similar kind of experiment 00:01:54.870 --> 00:01:58.080 in the plasma membrane, do you remember 00:01:58.080 --> 00:02:01.460 what I said about the levels of cholesterol? 00:02:01.460 --> 00:02:04.650 So they distributed throughout the cell, in all membranes. 00:02:04.650 --> 00:02:06.330 Where is the most cholesterol? 00:02:09.490 --> 00:02:12.360 So if you don't remember, it's the plasma membrane. 00:02:12.360 --> 00:02:17.490 So say, instead of having 7% or 8% of the lipids cholesterol, 00:02:17.490 --> 00:02:20.460 say you had 40%-- 00:02:20.460 --> 00:02:22.410 that's an over-exaggeration-- do you 00:02:22.410 --> 00:02:26.360 think this kind of an experiment would be hard to do, 00:02:26.360 --> 00:02:30.380 that they've talked about in this paper? 00:02:30.380 --> 00:02:32.190 So you would want to do this-- if you 00:02:32.190 --> 00:02:34.785 tried to do the same experiment with the plasma membrane? 00:02:43.444 --> 00:02:45.360 So the key issue that you need to think about, 00:02:45.360 --> 00:02:48.030 is go back and look at the changes-- 00:02:48.030 --> 00:02:50.950 they did a whole bunch of different experiments. 00:02:50.950 --> 00:02:53.070 The numbers are squishy, but they came up 00:02:53.070 --> 00:02:56.250 with numbers that reproduced themselves, I thought, 00:02:56.250 --> 00:02:58.080 in an amazing way. 00:02:58.080 --> 00:02:59.990 But now say you wanted to do this 00:02:59.990 --> 00:03:06.090 in the plasma membrane, where the levels of cholesterol 00:03:06.090 --> 00:03:06.780 are much higher. 00:03:11.280 --> 00:03:13.150 Do you think it would be easy to do? 00:03:13.150 --> 00:03:15.440 Using the same tech techniques that 00:03:15.440 --> 00:03:19.470 are described, that we're going to discuss, or not? 00:03:19.470 --> 00:03:22.640 And what would the issues be? 00:03:22.640 --> 00:03:23.380 Yeah? 00:03:23.380 --> 00:03:25.426 AUDIENCE: So they had to deplete the cholesterol 00:03:25.426 --> 00:03:28.640 from the membrane, and so that would probably 00:03:28.640 --> 00:03:32.545 be hard to deplete it to a level that's low enough, so that you 00:03:32.545 --> 00:03:34.150 don't get the activity. 00:03:34.150 --> 00:03:34.651 Right? 00:03:34.651 --> 00:03:35.983 JOANNE STUBBE: So, I don't know. 00:03:35.983 --> 00:03:37.560 So that's an interesting question. 00:03:37.560 --> 00:03:38.870 So you'd have to deplete-- 00:03:38.870 --> 00:03:40.411 so that's going to be it, we're going 00:03:40.411 --> 00:03:42.710 to have to control the cholesterol levels. 00:03:42.710 --> 00:03:44.900 But what change-- if you looked at the changes 00:03:44.900 --> 00:03:48.665 in levels of cholesterol in the ER, how much did they change? 00:03:56.280 --> 00:03:57.760 They change from what to what? 00:03:57.760 --> 00:04:03.820 From-- 2% to 7%. 00:04:03.820 --> 00:04:07.120 Say that you were in that same range of change that 00:04:07.120 --> 00:04:12.370 was going to turn on a switch in the plasma membrane. 00:04:12.370 --> 00:04:14.500 And say you could control the levels. 00:04:14.500 --> 00:04:18.550 Do you think it would be easy to see that? 00:04:18.550 --> 00:04:23.762 So you start with 40%, say, that's the norm. 00:04:23.762 --> 00:04:26.910 Say the change was very similar to what 00:04:26.910 --> 00:04:28.760 you see in the change in the ER-- 00:04:28.760 --> 00:04:30.510 do you think that would be easy to detect? 00:04:30.510 --> 00:04:33.660 No, because now you have two big numbers, 00:04:33.660 --> 00:04:36.140 and there's a huge amount of error 00:04:36.140 --> 00:04:37.530 in this method of analysis. 00:04:37.530 --> 00:04:39.321 So those are the kinds of things I'm trying 00:04:39.321 --> 00:04:41.350 to get you to think about. 00:04:41.350 --> 00:04:43.800 I don't know why it's the ER-- 00:04:43.800 --> 00:04:46.470 I mean, everybody's focused on the ER. 00:04:46.470 --> 00:04:50.160 Could cholesterol and other organelles 00:04:50.160 --> 00:04:52.200 have a different regulatory mechanism? 00:04:52.200 --> 00:04:55.650 Or somehow be connected, still, to what's going on in the ER? 00:04:55.650 --> 00:04:59.880 Could be-- I mean, you start out with the simplest model 00:04:59.880 --> 00:05:03.450 you can get and you test it, but then as you learn more, 00:05:03.450 --> 00:05:05.910 or we have more and more technology, 00:05:05.910 --> 00:05:08.880 we learn new things, you go back and you revisit and rethink 00:05:08.880 --> 00:05:11.890 about what's going on. 00:05:11.890 --> 00:05:13.950 So the key question is, it's really 00:05:13.950 --> 00:05:17.430 this switch of having cholesterol 00:05:17.430 --> 00:05:20.866 that keeps it in the membrane, or not having cholesterol. 00:05:20.866 --> 00:05:22.740 And the question is, what are the differences 00:05:22.740 --> 00:05:28.740 in the levels that allow turn on of cholesterol-- biosynthesis 00:05:28.740 --> 00:05:34.950 and LDL biosynthesis, which then allows uptake of cholesterol 00:05:34.950 --> 00:05:35.790 from the diet? 00:05:35.790 --> 00:05:38.422 OK, so that's the question. 00:05:38.422 --> 00:05:39.630 And what does this look like? 00:05:39.630 --> 00:05:42.080 And people hadn't measured this by any method, 00:05:42.080 --> 00:05:45.480 and this model I've gone through a number of times in class 00:05:45.480 --> 00:05:47.520 today, so I'm not going to go through it again. 00:05:47.520 --> 00:05:49.830 Hopefully you all know that in some form in your head, 00:05:49.830 --> 00:05:53.870 or you have the picture in front of you so you can remember it. 00:05:53.870 --> 00:05:55.620 So these are the questions I want to pose, 00:05:55.620 --> 00:05:58.470 and I want you guys to do the talking today. 00:05:58.470 --> 00:06:02.980 And what I'm going to do is, I have most of the figures 00:06:02.980 --> 00:06:06.380 on my PowerPoint, so we can bring them up and look at them. 00:06:06.380 --> 00:06:08.110 And you can tell me what you see. 00:06:08.110 --> 00:06:13.070 And then everybody might be seeing something different-- 00:06:13.070 --> 00:06:16.380 and so we're thinking about this differently, 00:06:16.380 --> 00:06:20.190 and maybe we come to some kind of consensus about 00:06:20.190 --> 00:06:23.200 whether these experiments were carried out well or not. 00:06:23.200 --> 00:06:25.920 So one of the first things-- so these 00:06:25.920 --> 00:06:28.470 will be the general things, and then we'll step through them. 00:06:28.470 --> 00:06:33.150 But they wanted to perturb the cellular cholesterol levels. 00:06:33.150 --> 00:06:36.620 And how did they end up doing that? 00:06:36.620 --> 00:06:38.190 Did that make sense? 00:06:38.190 --> 00:06:40.360 We talked a little bit about this already. 00:06:40.360 --> 00:06:42.720 I mean, what did they use as tools to do that? 00:06:45.648 --> 00:06:47.469 AUDIENCE: [INAUDIBLE] 00:06:47.469 --> 00:06:49.260 JOANNE STUBBE: So you need to speak louder, 00:06:49.260 --> 00:06:50.301 because I really am deaf. 00:06:50.301 --> 00:06:52.230 Sorry. 00:06:52.230 --> 00:06:53.744 AUDIENCE: So just, right here, they 00:06:53.744 --> 00:06:56.160 were careful of the amount of cholesterol present in this? 00:06:56.160 --> 00:06:57.618 JOANNE STUBBE: So that's one place, 00:06:57.618 --> 00:06:59.550 so they can deplete cholesterol for the media. 00:06:59.550 --> 00:07:02.670 But then what did they do? 00:07:02.670 --> 00:07:04.800 So the whole paper is about this-- how did they 00:07:04.800 --> 00:07:06.030 control the [INAUDIBLE]? 00:07:06.030 --> 00:07:08.490 Let's assume that they can do that, 00:07:08.490 --> 00:07:09.820 and they got good at that. 00:07:09.820 --> 00:07:11.760 I think a lot of people have used that method, 00:07:11.760 --> 00:07:15.450 and so they can deplete media. 00:07:15.450 --> 00:07:20.340 So how did they deplete cholesterol? 00:07:20.340 --> 00:07:23.460 There was some unusual ways to deplete cholesterol 00:07:23.460 --> 00:07:24.690 in this paper. 00:07:24.690 --> 00:07:26.746 Did any of you pick up on that? 00:07:26.746 --> 00:07:29.520 AUDIENCE: A chemical that could bind to cholesterol. 00:07:29.520 --> 00:07:32.100 JOANNE STUBBE: So did you think that was unusual? 00:07:32.100 --> 00:07:34.150 Did any of you look up what that was? 00:07:34.150 --> 00:07:37.695 AUDIENCE: It was a kind of carbohydrate 00:07:37.695 --> 00:07:40.056 that can bind to cholesterol. 00:07:40.056 --> 00:07:42.560 JOANNE STUBBE: Yeah, so but what was interesting about it, 00:07:42.560 --> 00:07:45.890 it was hydroxypropyl-- 00:07:45.890 --> 00:07:48.570 remember HP, cyclodextrin. 00:07:48.570 --> 00:07:50.310 We're going to look at this in a minute. 00:07:50.310 --> 00:07:52.110 But what do we know-- 00:07:52.110 --> 00:07:57.380 what was the other molecule they used to add cholesterol back? 00:07:57.380 --> 00:08:01.110 AUDIENCE: Another form of that molecule is-- 00:08:01.110 --> 00:08:03.871 JOANNE STUBBE: So methyl-cyclodextrin-- 00:08:03.871 --> 00:08:05.370 I'm going to show you the structure, 00:08:05.370 --> 00:08:07.450 but they aren't very different. 00:08:07.450 --> 00:08:12.750 So have any of you ever heard of cyclodextrin before? 00:08:12.750 --> 00:08:16.170 People won the Nobel Prize for that, Don Cram won it, 00:08:16.170 --> 00:08:19.219 Breslow spent his whole life studying host guest 00:08:19.219 --> 00:08:19.760 interactions. 00:08:19.760 --> 00:08:23.700 So you guys, I don't know what you teach you now anymore, 00:08:23.700 --> 00:08:26.400 but that used to be something that was taught a lot, 00:08:26.400 --> 00:08:28.320 host guest interactions, trying to understand 00:08:28.320 --> 00:08:31.875 weak non-covalent interactions as the basis for understanding 00:08:31.875 --> 00:08:33.150 catalysis. 00:08:33.150 --> 00:08:34.590 But to me, that was-- 00:08:34.590 --> 00:08:36.990 immediately when I saw this, what the heck's going on? 00:08:36.990 --> 00:08:39.740 So then I Googled it, and immediately-- 00:08:39.740 --> 00:08:42.780 and I don't know anything about hydroxypropyl-- you Google it, 00:08:42.780 --> 00:08:43.919 you look it up. 00:08:43.919 --> 00:08:47.040 And then you look at it, and if you were a chemist 00:08:47.040 --> 00:08:49.980 and you were really interested in the molecular interactions, 00:08:49.980 --> 00:08:52.000 you might make a model of it. 00:08:52.000 --> 00:08:55.860 And then see, what is the difference between that one 00:08:55.860 --> 00:08:58.620 little group, when you look at the structure, it's amazing. 00:08:58.620 --> 00:09:02.670 And that's the basis of most of the experiments. 00:09:02.670 --> 00:09:06.240 So you need to believe that they figured that out. 00:09:06.240 --> 00:09:10.570 And that's not in this paper, so if you really cared about it 00:09:10.570 --> 00:09:12.720 you would have to go back and read earlier papers, 00:09:12.720 --> 00:09:16.470 and see what are the experiments that led them 00:09:16.470 --> 00:09:19.050 to focus on these molecules? 00:09:19.050 --> 00:09:23.430 How else did they end up getting cholesterol levels back 00:09:23.430 --> 00:09:24.060 into the cell? 00:09:24.060 --> 00:09:26.684 Do you remember what the other method was? 00:09:26.684 --> 00:09:29.100 So we'll come back and we'll talk about this in a minute-- 00:09:29.100 --> 00:09:30.391 so that was one of the methods. 00:09:35.760 --> 00:09:37.767 AUDIENCE: They added two kind of sterols. 00:09:37.767 --> 00:09:40.100 JOANNE STUBBE: OK, so they did add two kind of sterols-- 00:09:40.100 --> 00:09:41.516 and they tried to figure out, this 00:09:41.516 --> 00:09:45.200 is another unknown, what was the difference between the sterols? 00:09:45.200 --> 00:09:47.030 Simply a hydroxyl group. 00:09:47.030 --> 00:09:53.020 OK, so if you looked at this, cholesterol is this guy. 00:09:53.020 --> 00:09:57.080 And then they had something like this guy-- 00:09:57.080 --> 00:09:59.750 25, and remember where [INAUDIBLE] the side chain, 00:09:59.750 --> 00:10:06.080 hanging out of the little [? cheer ?] system you have. 00:10:06.080 --> 00:10:08.100 I don't think they learned very much from that. 00:10:08.100 --> 00:10:09.980 And in fact, in your problem set, 00:10:09.980 --> 00:10:14.630 you had all of these different cholesterol analogs. 00:10:14.630 --> 00:10:17.060 I mean, I think we still really don't get it. 00:10:17.060 --> 00:10:19.320 That's complicated-- we talked about this in class. 00:10:19.320 --> 00:10:21.830 You have these transmembrane helices-- 00:10:21.830 --> 00:10:25.310 what is it that's actually the signaling agent? 00:10:25.310 --> 00:10:28.670 So people are still asking that question, 00:10:28.670 --> 00:10:32.460 and we haven't quite gotten that far. 00:10:32.460 --> 00:10:38.480 But if you've read the reading, for HMG CoA reductase 00:10:38.480 --> 00:10:40.820 degradation, which is what we we're 00:10:40.820 --> 00:10:42.350 going to be talking about in class, 00:10:42.350 --> 00:10:47.600 the signaler is not the sterile, it's lanosterol. 00:10:47.600 --> 00:10:49.520 OK, and where have you seen lanosterol? 00:10:49.520 --> 00:10:52.940 The biosynthetic pathway has lanosterol 00:10:52.940 --> 00:10:54.270 sitting in the middle. 00:10:54.270 --> 00:10:58.130 It's not all that different, structurally, from cholesterol. 00:10:58.130 --> 00:11:01.760 You need to go back in, they all have four-membered rings, 00:11:01.760 --> 00:11:06.060 they have different extra methyl groups. 00:11:06.060 --> 00:11:08.900 So people are trying to sort that out. 00:11:08.900 --> 00:11:10.580 I don't think we really know. 00:11:10.580 --> 00:11:11.150 But how well? 00:11:11.150 --> 00:11:13.954 So you're right, they use sterols. 00:11:13.954 --> 00:11:16.370 They didn't use that, they didn't see very much difference 00:11:16.370 --> 00:11:17.120 with the sterols. 00:11:17.120 --> 00:11:20.450 What was the other way, which is sort of unusual, 00:11:20.450 --> 00:11:25.010 that they added cholesterol back into the system. 00:11:25.010 --> 00:11:29.660 So they could add it back with the methyl cyclodextrin-- 00:11:29.660 --> 00:11:32.380 they told you that that worked, and if you believe that-- 00:11:32.380 --> 00:11:35.438 and you look at the data-- it looked like that was happening. 00:11:39.920 --> 00:11:41.090 Nobody remembers? 00:11:41.090 --> 00:11:44.890 OK, well, we'll get to that in a little bit. 00:11:44.890 --> 00:11:47.720 OK, so the question we're focusing on 00:11:47.720 --> 00:11:50.570 is what are the changes in concentrations 00:11:50.570 --> 00:11:53.030 of cholesterol in the ER? 00:11:53.030 --> 00:11:58.520 So what method did they use to try 00:11:58.520 --> 00:12:03.320 to separate the ER membranes from all the other membranes? 00:12:05.948 --> 00:12:08.510 AUDIENCE: They first separated the [INAUDIBLE]---- 00:12:08.510 --> 00:12:10.804 JOANNE STUBBE: They separated the what? 00:12:10.804 --> 00:12:15.524 AUDIENCE: The sterols and the nucleus in the [INAUDIBLE].. 00:12:15.524 --> 00:12:16.940 JOANNE STUBBE: OK, so that's good. 00:12:16.940 --> 00:12:18.900 You can separate out the nucleus, 00:12:18.900 --> 00:12:21.920 and you could do that by ultracentrifugation-- we've 00:12:21.920 --> 00:12:25.520 seen that used in different kinds of ultracentrifugation. 00:12:25.520 --> 00:12:30.600 We've seen the different particles, 00:12:30.600 --> 00:12:36.290 the lipoproteins in the diet, how do we separate those? 00:12:36.290 --> 00:12:38.462 We talked about that in class briefly, 00:12:38.462 --> 00:12:39.920 you haven't had any papers to read. 00:12:39.920 --> 00:12:42.110 But what was the method of separation? 00:12:42.110 --> 00:12:43.800 If you look at all those particles-- 00:12:43.800 --> 00:12:46.820 remember we had a little cartoon of all the particles, 00:12:46.820 --> 00:12:50.400 and we focused on LDL, which is the particle that 00:12:50.400 --> 00:12:51.690 has the most cholesterol. 00:12:51.690 --> 00:12:53.960 So that's why everybody is focusing on that. 00:12:53.960 --> 00:12:55.970 What was the basis of the separation? 00:12:55.970 --> 00:12:57.730 AUDIENCE: Was it sucrose screening? 00:12:57.730 --> 00:12:59.420 JOANNE STUBBE: Was the what? 00:12:59.420 --> 00:13:00.260 AUDIENCE: Was it a sucrose screening-- 00:13:00.260 --> 00:13:01.100 the ultracentrifugation? 00:13:01.100 --> 00:13:02.266 JOANNE STUBBE: You need to-- 00:13:02.266 --> 00:13:04.200 AUDIENCE: Did they use a sucrose screening, 00:13:04.200 --> 00:13:05.532 like ultracentrifugation? 00:13:05.532 --> 00:13:07.240 JOANNE STUBBE: Yeah, ultracentrifugation. 00:13:07.240 --> 00:13:09.250 But how did the-- 00:13:09.250 --> 00:13:10.750 AUDIENCE: For the sucrose screening? 00:13:10.750 --> 00:13:13.041 JOANNE STUBBE: Yeah, OK, so they have different density 00:13:13.041 --> 00:13:15.520 gradients. , OK so that's going to be a key thing, 00:13:15.520 --> 00:13:18.084 and that's because if you look at the composition, 00:13:18.084 --> 00:13:19.750 they have different amounts of proteins, 00:13:19.750 --> 00:13:21.250 different amounts of fats. 00:13:21.250 --> 00:13:23.930 And they have different-- they float differently. 00:13:23.930 --> 00:13:27.520 So that's the method that they're going to use here. 00:13:27.520 --> 00:13:28.510 Is that a good method? 00:13:28.510 --> 00:13:30.810 Can you think of a better method? 00:13:30.810 --> 00:13:35.690 So in order to understand the switch for cholesterol, 00:13:35.690 --> 00:13:39.130 you've got to be able to measure the changes in cholesterol. 00:13:39.130 --> 00:13:42.160 Not an easy problem, because cholesterol is 00:13:42.160 --> 00:13:45.520 really insoluble in everything. 00:13:45.520 --> 00:13:48.190 And so how much is really in there, 00:13:48.190 --> 00:13:51.330 and how does it change under different sets of conditions? 00:13:51.330 --> 00:13:53.020 So is this a good method? 00:13:53.020 --> 00:13:53.830 What do you think? 00:13:53.830 --> 00:13:56.526 We'll look at the method in a little more detail, when 00:13:56.526 --> 00:13:58.150 I pull up the figures, but what did you 00:13:58.150 --> 00:14:01.540 think when you read the paper? 00:14:04.991 --> 00:14:07.456 AUDIENCE: Seems a pretty good method, 00:14:07.456 --> 00:14:12.386 other than that they're slightly different 00:14:12.386 --> 00:14:20.128 any other like properties different from the membrane 00:14:20.128 --> 00:14:21.753 than say, press on golgi bodies and ER. 00:14:21.753 --> 00:14:23.750 So it's like the only one I can think of. 00:14:23.750 --> 00:14:26.460 JOANNE STUBBE: Yeah, so the question is, you 00:14:26.460 --> 00:14:28.230 could you separate? 00:14:28.230 --> 00:14:32.660 Even separating the nucleus from the cytosol is not so trivial. 00:14:32.660 --> 00:14:35.010 But these methods are really gross methods, 00:14:35.010 --> 00:14:39.120 and during the centrifugation, things diffuse. 00:14:39.120 --> 00:14:40.890 So if you're having close separations, 00:14:40.890 --> 00:14:43.420 it's a equlibrating down this thing. 00:14:43.420 --> 00:14:46.070 And so you're getting your proteins, 00:14:46.070 --> 00:14:48.420 or your lipids are spreading out. 00:14:48.420 --> 00:14:52.680 Is there anything else any of you experience with insoluble-- 00:14:52.680 --> 00:14:55.770 this is what we're dealing with, is an insoluble mess, 00:14:55.770 --> 00:15:00.150 and how do you how do you separate things in a way 00:15:00.150 --> 00:15:02.190 that you have control over it so that you 00:15:02.190 --> 00:15:04.830 can address the key questions in this paper? 00:15:09.810 --> 00:15:12.170 Nobody thought about anything else? 00:15:12.170 --> 00:15:14.300 Did you like this method? 00:15:14.300 --> 00:15:17.862 Were you convinced by the data? 00:15:17.862 --> 00:15:20.352 AUDIENCE: I mean, like I couldn't necessarily 00:15:20.352 --> 00:15:22.842 think of something better. 00:15:22.842 --> 00:15:25.830 I don't know, I guess the thing that 00:15:25.830 --> 00:15:29.814 sketches me out the most about it just like how-- 00:15:29.814 --> 00:15:32.215 I'm not really familiar with the method. 00:15:32.215 --> 00:15:33.798 I haven't done this myself, so I don't 00:15:33.798 --> 00:15:37.734 know how that process affects the membrane integrity. 00:15:37.734 --> 00:15:40.150 JOANNE STUBBE: So that's an incredibly important question, 00:15:40.150 --> 00:15:42.880 because lipids confuse. 00:15:42.880 --> 00:15:43.879 They can mix. 00:15:43.879 --> 00:15:46.420 The question is, what are the rate constants for all of that? 00:15:46.420 --> 00:15:48.640 And we don't really teach very much 00:15:48.640 --> 00:15:50.740 in the introductory courses about lipids, 00:15:50.740 --> 00:15:55.060 and they're partitioning between other membranes and fusion, 00:15:55.060 --> 00:15:55.910 and all that stuff. 00:15:55.910 --> 00:15:58.780 But if you think about it, that's what the cell is, right? 00:15:58.780 --> 00:16:00.670 How do you get a plasma membrane, 00:16:00.670 --> 00:16:03.530 and all these membranes around all these little organelles-- 00:16:03.530 --> 00:16:06.100 that's an amazing observation. 00:16:06.100 --> 00:16:08.200 And we've seen in class already, what 00:16:08.200 --> 00:16:13.990 have we seen to get LDL receptor from here to the plasma 00:16:13.990 --> 00:16:15.090 membrane? 00:16:15.090 --> 00:16:17.710 How do we have to do that? 00:16:17.710 --> 00:16:20.770 We had to use these little vesicles. 00:16:20.770 --> 00:16:24.020 So you're generating something over here, 00:16:24.020 --> 00:16:26.310 it goes through the Golgi stack. 00:16:26.310 --> 00:16:28.540 Again, another set of membranes has 00:16:28.540 --> 00:16:31.810 got to come out the different levels of the Golgi stack. 00:16:31.810 --> 00:16:37.360 And then it's still got to get into the plasma membrane, 00:16:37.360 --> 00:16:39.730 and fuse, and dump its cargo. 00:16:39.730 --> 00:16:42.610 So I think it's an amazing process. 00:16:42.610 --> 00:16:44.110 And people interested in evolution, 00:16:44.110 --> 00:16:46.630 this is one of the major things people are focused on 00:16:46.630 --> 00:16:51.760 is, how can you make cells, little fake cells, 00:16:51.760 --> 00:16:53.950 artificial cells, that can replicate themselves. 00:16:53.950 --> 00:16:55.366 You can make it, and they're going 00:16:55.366 --> 00:16:57.820 to have to divide and fuse. 00:16:57.820 --> 00:17:00.520 And it's exactly the same problem here. 00:17:00.520 --> 00:17:04.569 And so this question of fluidity is an extremely important 00:17:04.569 --> 00:17:05.079 question. 00:17:05.079 --> 00:17:07.960 And a lot of people that focus on lipids-- 00:17:07.960 --> 00:17:12.619 which is not a popular thing to study, because it's so hard-- 00:17:12.619 --> 00:17:14.170 it's incredibly important. 00:17:14.170 --> 00:17:17.410 And people that look at membrane proteins, 00:17:17.410 --> 00:17:20.440 they almost always have lipids on them. 00:17:20.440 --> 00:17:22.060 And when you do them yourself, you 00:17:22.060 --> 00:17:25.300 have a detergent, which is not a real lipid-- 00:17:25.300 --> 00:17:26.660 does that change the property? 00:17:26.660 --> 00:17:28.630 So all of these questions, I think, 00:17:28.630 --> 00:17:32.110 are really central to what happens 00:17:32.110 --> 00:17:36.860 in the membranes, which is a lot of stuff inside the cell. 00:17:36.860 --> 00:17:40.000 So I think it's good to question what they did. 00:17:40.000 --> 00:17:43.730 I think their results turned out to be quite interesting. 00:17:43.730 --> 00:17:45.040 But we'll come back-- 00:17:45.040 --> 00:17:46.840 I think that was a hard problem. 00:17:46.840 --> 00:17:49.340 And so we'll come back and we'll look at this. 00:17:49.340 --> 00:17:56.050 And so then, let's say that we could end up separating things. 00:17:56.050 --> 00:18:01.390 Then the question is, what was the key type of measurement 00:18:01.390 --> 00:18:07.600 they made, where they could correlate the changes 00:18:07.600 --> 00:18:08.800 in cholesterol levels-- 00:18:08.800 --> 00:18:11.470 we talked about, you can control perhaps the cholesterol 00:18:11.470 --> 00:18:14.970 levels with the cyclodextrin. 00:18:14.970 --> 00:18:18.310 But then, how did they correlate the changes 00:18:18.310 --> 00:18:20.820 in the cholesterol levels in the membrane 00:18:20.820 --> 00:18:25.570 with this transcriptional regulation? 00:18:25.570 --> 00:18:30.310 Which, that is what happens with the steroid-responsive 00:18:30.310 --> 00:18:32.870 element-binding protein, the transcription factor. 00:18:32.870 --> 00:18:36.350 So what happens in that process? 00:18:36.350 --> 00:18:41.770 What are the changes in the SRE BP 00:18:41.770 --> 00:18:45.610 dependent on the concentrations of the cholesterol? 00:18:45.610 --> 00:18:48.150 And how did they take advantage of that 00:18:48.150 --> 00:18:53.830 in answering this question about what the cholesterol 00:18:53.830 --> 00:18:58.870 levels were that allowed you to turn on transcription 00:18:58.870 --> 00:19:03.440 of LDL receptor, and HMG CoA reductase. 00:19:03.440 --> 00:19:04.750 So what's the major assay? 00:19:04.750 --> 00:19:06.937 We'll look at that, as well. 00:19:06.937 --> 00:19:08.770 So if you go back and you look at the model, 00:19:08.770 --> 00:19:09.985 what happens in this model? 00:19:15.170 --> 00:19:17.050 All right, here we go-- 00:19:17.050 --> 00:19:18.610 what happens in this model? 00:19:18.610 --> 00:19:20.665 What's happening to SREBP? 00:19:23.395 --> 00:19:25.375 AUDIENCE: It has completely changed 00:19:25.375 --> 00:19:28.345 and exposed [INAUDIBLE]. 00:19:28.345 --> 00:19:30.700 JOANNE STUBBE: No, that's SCAP-- 00:19:30.700 --> 00:19:33.140 SCAP, that's this guy. 00:19:33.140 --> 00:19:33.970 OK? 00:19:33.970 --> 00:19:35.714 So SCAP, that's a key player. 00:19:35.714 --> 00:19:36.880 That's what we talked about. 00:19:36.880 --> 00:19:38.890 I know the names are all confusing. 00:19:38.890 --> 00:19:41.260 You're going to need to write these down to remember. 00:19:41.260 --> 00:19:42.920 The names are very confusing. 00:19:42.920 --> 00:19:43.653 Yeah? 00:19:43.653 --> 00:19:48.090 AUDIENCE: So the SCAP SREBP, whatever you call it, 00:19:48.090 --> 00:19:54.840 complex move signal g-apperatus then part of it's cleaved 00:19:54.840 --> 00:19:56.120 and moves to the nucleus? 00:19:56.120 --> 00:19:58.150 JOANNE STUBBE: Right, so how could you 00:19:58.150 --> 00:19:59.710 take advantage of that? 00:19:59.710 --> 00:20:03.700 This is the key observation that they're taking advantage of, 00:20:03.700 --> 00:20:05.140 to ask the question-- 00:20:05.140 --> 00:20:08.740 since this whole process is dependent on the concentration 00:20:08.740 --> 00:20:09.750 of cholesterol. 00:20:09.750 --> 00:20:11.320 If you have high cholesterol, there's 00:20:11.320 --> 00:20:14.650 no way you want this to happen-- you want to shut it off. 00:20:14.650 --> 00:20:19.060 If you have low cholesterol, you want to turn these guys on. 00:20:19.060 --> 00:20:22.480 So this movement is the key. 00:20:22.480 --> 00:20:24.460 And what do we see, if we look at what 00:20:24.460 --> 00:20:27.467 happens to this protein, SREBP, what happens to it 00:20:27.467 --> 00:20:28.300 during this process? 00:20:30.970 --> 00:20:31.830 It gets cleaved. 00:20:31.830 --> 00:20:34.990 And how could you monitor that cleavage? 00:20:34.990 --> 00:20:37.218 How do they do it in the paper? 00:20:37.218 --> 00:20:40.122 AUDIENCE: They used a-- 00:20:40.122 --> 00:20:42.574 was it a [? florifor-- ?] or is that the homework? 00:20:42.574 --> 00:20:44.740 JOANNE STUBBE: They could use a [? florifor, ?] they 00:20:44.740 --> 00:20:46.580 didn't do that. 00:20:46.580 --> 00:20:47.610 They did a what? 00:20:50.508 --> 00:20:58.154 AUDIENCE: They were able to separate the [INAUDIBLE] gel? 00:20:58.154 --> 00:21:00.070 JOANNE STUBBE: So it can be operated by a gel. 00:21:00.070 --> 00:21:03.840 So to me, this is quite an easy assay. 00:21:03.840 --> 00:21:05.100 Because if you look at this-- 00:21:05.100 --> 00:21:07.050 I don't remember what the molecular weight is, 00:21:07.050 --> 00:21:10.180 but it's a lot smaller over here. 00:21:10.180 --> 00:21:14.100 And so, that turns out to be a great assay. 00:21:14.100 --> 00:21:18.630 So that part of their analysis, I think, 00:21:18.630 --> 00:21:21.100 was a really smart part of the analysis. 00:21:21.100 --> 00:21:23.620 And so then the question becomes, 00:21:23.620 --> 00:21:26.320 can you quantitate all of this? 00:21:26.320 --> 00:21:28.680 So if you have a lot of cholesterol, 00:21:28.680 --> 00:21:29.790 this doesn't happen. 00:21:29.790 --> 00:21:35.100 And so everything is bigger, and resides in the membrane. 00:21:35.100 --> 00:21:37.420 You could even probably look at that. 00:21:37.420 --> 00:21:43.350 Whereas, when the cholesterol is really lower, things go there. 00:21:43.350 --> 00:21:44.730 And it's everything in between. 00:21:44.730 --> 00:21:46.660 The question is, what is the concept-- 00:21:46.660 --> 00:21:50.820 can you measure if you have X% cholesterol in the ER, 00:21:50.820 --> 00:21:55.620 how much do you have to decrease it to see a change or a switch 00:21:55.620 --> 00:21:58.260 in where this protein goes? 00:21:58.260 --> 00:22:02.070 So I think the experimental design is actually 00:22:02.070 --> 00:22:04.320 amazingly creative. 00:22:04.320 --> 00:22:07.990 But then you see the data of the other side. 00:22:07.990 --> 00:22:13.680 And what I want to do now is focus on what the issues are. 00:22:13.680 --> 00:22:17.640 So we're going to come back and look at, 00:22:17.640 --> 00:22:19.920 how did they look at SREBP? 00:22:19.920 --> 00:22:23.040 So you could look at this a number 00:22:23.040 --> 00:22:28.280 of ways-- you could look at this by protein gel directly. 00:22:28.280 --> 00:22:32.040 How else do people look at proteins using westerns? 00:22:32.040 --> 00:22:34.560 What's a western? 00:22:34.560 --> 00:22:37.165 Anybody know what a western analysis is? 00:22:37.165 --> 00:22:39.165 Didn't I ask you that at the beginning of class? 00:22:42.430 --> 00:22:44.170 How else do you detect proteins? 00:22:44.170 --> 00:22:48.020 You've seen this in the first half of the semester a lot. 00:22:48.020 --> 00:22:49.130 Yeah, antibodies. 00:22:49.130 --> 00:22:51.630 So if you have antibodies to this-- 00:22:51.630 --> 00:22:54.670 and we'll talk about this, because the western analysis, 00:22:54.670 --> 00:22:58.180 which people use all the time, and there are so 00:22:58.180 --> 00:22:59.860 many issues with it, that I think 00:22:59.860 --> 00:23:03.850 I want you to think about what the issues are. 00:23:03.850 --> 00:23:06.400 And then you correlate the two-- 00:23:06.400 --> 00:23:07.960 changing the levels of cholesterol. 00:23:07.960 --> 00:23:12.340 Which they measure by mass spec after separation 00:23:12.340 --> 00:23:15.550 and purification of lipids, and the cleavage. 00:23:15.550 --> 00:23:17.920 And they plot the data, and that's 00:23:17.920 --> 00:23:20.500 where they got the analysis from. 00:23:20.500 --> 00:23:23.140 So the first thing that you want to do-- the first thing, 00:23:23.140 --> 00:23:27.370 and the key to everything, is separation of the membranes. 00:23:27.370 --> 00:23:31.960 And so, this is a cartoon of when you put something, 00:23:31.960 --> 00:23:35.070 you load something on the top, and you have a gradient, 00:23:35.070 --> 00:23:37.630 and the gradient could be made of a number of things. 00:23:37.630 --> 00:23:40.500 Have any of you ever run these kinds of gradients? 00:23:40.500 --> 00:23:43.000 OK, so you can make them out of glycerol, 00:23:43.000 --> 00:23:44.530 you can make them out of sucrose-- 00:23:44.530 --> 00:23:46.840 did anybody look at how these gradients were made? 00:23:46.840 --> 00:23:48.814 Did you read the experimental carefully enough 00:23:48.814 --> 00:23:49.480 to look at that? 00:23:54.720 --> 00:23:56.605 Yeah, how do you make a sucrose gradient? 00:24:02.790 --> 00:24:03.670 You have no idea? 00:24:03.670 --> 00:24:06.050 But yeah, so layering. 00:24:06.050 --> 00:24:08.709 So what you really like to do is have a continuous gradient, 00:24:08.709 --> 00:24:09.250 or something. 00:24:09.250 --> 00:24:11.920 But sucrose is incredibly viscous. 00:24:11.920 --> 00:24:14.020 So if you were trying to make a linear gradient, 00:24:14.020 --> 00:24:16.122 which you could do by mixing two things 00:24:16.122 --> 00:24:18.580 of different concentrations-- if you could get them to stir 00:24:18.580 --> 00:24:20.947 really well, and then add it in, and you 00:24:20.947 --> 00:24:22.030 could generate a gradient. 00:24:22.030 --> 00:24:24.850 But it's so hard to do, that what happens 00:24:24.850 --> 00:24:26.050 is they end up layering it. 00:24:26.050 --> 00:24:31.150 So they make X%, Y%, Z%, they put it down. 00:24:31.150 --> 00:24:33.610 And then they try to layer something on top of it. 00:24:33.610 --> 00:24:38.170 And then they put whatever the interest in at the top, 00:24:38.170 --> 00:24:40.930 and then they centrifuge it. 00:24:40.930 --> 00:24:43.960 So what are the issues? 00:24:43.960 --> 00:24:48.484 Do you think this is what the gradient would look like? 00:24:48.484 --> 00:24:49.900 So what are the issues when you're 00:24:49.900 --> 00:24:54.090 doing this, when you layer it? 00:24:54.090 --> 00:24:56.050 And this is why the data-- 00:24:56.050 --> 00:24:58.410 which we'll talk about in a minute-- 00:24:58.410 --> 00:25:01.684 is the data, or part of the issue is this method. 00:25:01.684 --> 00:25:03.600 That's why you need to think about the method. 00:25:03.600 --> 00:25:05.370 And there are better ways to do this. 00:25:05.370 --> 00:25:09.000 And it really depends on what you're trying to separate. 00:25:09.000 --> 00:25:11.850 So if this band-- 00:25:11.850 --> 00:25:14.130 say these were two bands, you wouldn't really 00:25:14.130 --> 00:25:15.676 get very much separation at all. 00:25:15.676 --> 00:25:17.050 If there were two separate things 00:25:17.050 --> 00:25:21.870 that sedimented under these conditions very close together. 00:25:21.870 --> 00:25:24.460 So what would happen when you're sedimenting this? 00:25:24.460 --> 00:25:26.750 Does anybody have any idea how long it takes? 00:25:26.750 --> 00:25:29.040 Do you think you'd do this in a centrifuge, 00:25:29.040 --> 00:25:30.810 you spin it for three minutes, and then-- 00:25:33.460 --> 00:25:37.620 so sometimes you sediment these things for 16, 20 hours. 00:25:37.620 --> 00:25:41.790 So what happens during the sedimentation? 00:25:41.790 --> 00:25:44.520 That might make this more challenging, 00:25:44.520 --> 00:25:46.892 in terms of separating what you want to separate? 00:25:46.892 --> 00:25:49.100 AUDIENCE: I'm not sure, but it [INAUDIBLE] diffusion. 00:25:49.100 --> 00:25:51.450 JOANNE STUBBE: Yeah, so exactly, you have diffusion. 00:25:51.450 --> 00:25:54.330 And even when you've layered things on top of each other 00:25:54.330 --> 00:25:57.030 like that, you start to have diffusion. 00:25:57.030 --> 00:26:01.030 And if you shake up the tube a little bit, it's all over. 00:26:01.030 --> 00:26:03.720 So how do you prepare these things is not-- 00:26:03.720 --> 00:26:05.790 so people still use these methods, 00:26:05.790 --> 00:26:08.220 but I would like to see better methods. 00:26:08.220 --> 00:26:12.270 And so they tried one method with sucrose, 00:26:12.270 --> 00:26:14.130 and then that wasn't good enough. 00:26:14.130 --> 00:26:15.150 We'll look at the data. 00:26:15.150 --> 00:26:17.630 So they went to a second method. 00:26:17.630 --> 00:26:20.007 And where did they come up with this? 00:26:20.007 --> 00:26:21.840 I have no idea where they came up with this, 00:26:21.840 --> 00:26:24.840 but there was an MD PhD student in our class 00:26:24.840 --> 00:26:27.330 who had seen this and one of his classes, 00:26:27.330 --> 00:26:29.280 and they use it and some blood test. 00:26:29.280 --> 00:26:31.950 So I think that's probably where these guys got it from, 00:26:31.950 --> 00:26:35.310 because Brown and Goldstein are both MDs. 00:26:35.310 --> 00:26:38.220 But again, it's just another way to make a gradient. 00:26:38.220 --> 00:26:42.090 And I'm not sure why this gradient works 00:26:42.090 --> 00:26:45.960 as effectively as it does. 00:26:45.960 --> 00:26:48.520 But the first gradient didn't work so great, 00:26:48.520 --> 00:26:49.770 and we'll look at that data. 00:26:49.770 --> 00:26:52.290 So then they added on a few more steps, 00:26:52.290 --> 00:26:57.040 because they weren't happy with the level of separation. 00:26:57.040 --> 00:26:59.640 So looking at membranes, I think this 00:26:59.640 --> 00:27:02.070 is going to be more and more looking at membranes, 00:27:02.070 --> 00:27:04.910 because membranes, you have two leaflets-- 00:27:04.910 --> 00:27:07.020 the lipids and the leaflets are different. 00:27:07.020 --> 00:27:10.170 Do you think that affects the biology? 00:27:10.170 --> 00:27:12.870 I guarantee you it affects the biology in ways 00:27:12.870 --> 00:27:15.510 that we would really like to understand that I don't 00:27:15.510 --> 00:27:18.180 think we understand very well. 00:27:18.180 --> 00:27:20.910 When you isolate a membrane protein, have any of you 00:27:20.910 --> 00:27:24.280 ever isolated a membrane protein? 00:27:24.280 --> 00:27:25.950 So you have an insoluble-- 00:27:25.950 --> 00:27:28.945 it's in this lipid system. 00:27:28.945 --> 00:27:30.570 How do you think you get it out, so you 00:27:30.570 --> 00:27:34.680 can go through the steps, a protein purification 00:27:34.680 --> 00:27:37.680 that you've talked about, or you have probably done 00:27:37.680 --> 00:27:40.550 in an introductory lab course? 00:27:40.550 --> 00:27:42.360 What is the first thing you need to do? 00:27:49.122 --> 00:27:50.370 Yeah, solubalize it. 00:27:50.370 --> 00:27:51.995 And how do you solubalize it? 00:27:51.995 --> 00:27:53.120 AUDIENCE: With a detergent. 00:27:53.120 --> 00:27:55.161 JOANNE STUBBE: Yeah, with some kind of detergent. 00:27:55.161 --> 00:27:58.310 It's like what you saw with a kilo microns, or the bile acids 00:27:58.310 --> 00:27:59.640 that we talked about. 00:27:59.640 --> 00:28:01.700 So you can use different-- and people have 00:28:01.700 --> 00:28:03.500 their own favorite detergents. 00:28:03.500 --> 00:28:06.990 But again, that changes things. 00:28:06.990 --> 00:28:09.890 But otherwise, you can't purify anything 00:28:09.890 --> 00:28:13.120 unless you happen to have a membrane where 00:28:13.120 --> 00:28:14.739 the only protein in the membrane is 00:28:14.739 --> 00:28:17.030 the one you're interested in, which, of course, doesn't 00:28:17.030 --> 00:28:18.530 exist. 00:28:18.530 --> 00:28:20.090 So anyhow, they went through that. 00:28:20.090 --> 00:28:22.410 And then what did they end up seeing? 00:28:22.410 --> 00:28:25.430 So they went through different steps, 00:28:25.430 --> 00:28:32.941 and they separate them into different-- the supernate, 00:28:32.941 --> 00:28:36.590 or the light and the heavy membrane fractions. 00:28:36.590 --> 00:28:38.072 And then they have to analyze it. 00:28:38.072 --> 00:28:39.530 And so the question is, how do they 00:28:39.530 --> 00:28:45.230 analyze to tell how well these separations actually worked? 00:28:45.230 --> 00:28:49.930 What was the method that they did 00:28:49.930 --> 00:28:53.000 to determine whether they separated 00:28:53.000 --> 00:28:57.200 the ER from the plasma membrane, from the Golgi stacks, 00:28:57.200 --> 00:29:00.410 from the lisosomes, from the peroxisomes. 00:29:00.410 --> 00:29:03.320 So they have all we have all these little organelles 00:29:03.320 --> 00:29:04.130 in there. 00:29:04.130 --> 00:29:09.800 What did they do to test each one of these fractions? 00:29:09.800 --> 00:29:15.560 Let me ask you this question-- how do you think they got the-- 00:29:15.560 --> 00:29:20.360 how do you how did they get the material out of these gradients 00:29:20.360 --> 00:29:24.180 to do the experiments that I was just talking about. 00:29:24.180 --> 00:29:27.350 So they want to analyze what's in each of these bands. 00:29:27.350 --> 00:29:30.668 How did they get it out of this tube? 00:29:30.668 --> 00:29:32.419 AUDIENCE: Would they use a Pasteur filter? 00:29:32.419 --> 00:29:33.918 JOANNE STUBBE: So what do you think? 00:29:33.918 --> 00:29:35.890 You just stick it down in and suck it out? 00:29:35.890 --> 00:29:38.020 Well, I mean, yes, so what do you think? 00:29:38.020 --> 00:29:39.400 You could do that-- 00:29:39.400 --> 00:29:42.820 you open the top, you stick it in, you carefully stick it in. 00:29:42.820 --> 00:29:44.050 If you can see it. 00:29:44.050 --> 00:29:47.200 Lots of times you can see these lipids, because they're opaque, 00:29:47.200 --> 00:29:47.780 or something. 00:29:47.780 --> 00:29:48.580 So you can see. 00:29:48.580 --> 00:29:53.440 Or, if you still hope your sucrose layers, lots of times 00:29:53.440 --> 00:29:56.050 they layer in between the different concentrations 00:29:56.050 --> 00:29:59.170 of the sucrose, and you see white stuff precipitating. 00:29:59.170 --> 00:30:02.590 So you could conceivably stick a pipe head from the top 00:30:02.590 --> 00:30:03.940 and suck it out. 00:30:03.940 --> 00:30:05.410 AUDIENCE: But that would perturb all the other layers. 00:30:05.410 --> 00:30:06.250 JOANNE STUBBE: Absolutely it would 00:30:06.250 --> 00:30:07.526 perturb all the other layers. 00:30:07.526 --> 00:30:09.400 So here you're doing something-- it's already 00:30:09.400 --> 00:30:11.858 a very hard experiment, because they're all being perturbed 00:30:11.858 --> 00:30:14.190 anyhow, because of diffusion. 00:30:14.190 --> 00:30:16.450 So is there any other way you could think 00:30:16.450 --> 00:30:19.850 about separating these things? 00:30:19.850 --> 00:30:23.530 And so, the hint is that they use plastic tubes. 00:30:27.880 --> 00:30:29.760 So these things are not glass. 00:30:29.760 --> 00:30:30.820 Most centrifuges-- 00:30:30.820 --> 00:30:31.690 AUDIENCE: Freeze it? 00:30:31.690 --> 00:30:32.560 Cut it? 00:30:32.560 --> 00:30:35.540 JOANNE STUBBE: Well, so you don't do that, that could be-- 00:30:35.540 --> 00:30:37.320 OK, so you could. 00:30:37.320 --> 00:30:39.640 But you then have to, if you were cutting it, 00:30:39.640 --> 00:30:42.460 you still have to get it out of the tube. 00:30:42.460 --> 00:30:46.360 Unless you had a saw that didn't have any vibrations when 00:30:46.360 --> 00:30:49.270 you were cutting it, of course, which would not happen. 00:30:49.270 --> 00:30:52.690 But if you look here in this cartoon, 00:30:52.690 --> 00:30:54.700 so I gave you this, what are they doing here? 00:30:54.700 --> 00:30:58.390 They're sticking a syringe in through the side of the tube. 00:30:58.390 --> 00:31:00.530 And that's still what people use. 00:31:00.530 --> 00:31:03.240 So you can suck out-- if you can see something. 00:31:03.240 --> 00:31:05.170 So you have to be able to see in some way 00:31:05.170 --> 00:31:09.685 to know where to suck it out, so you might have a way, actually, 00:31:09.685 --> 00:31:12.250 in doing ultracentrifugations. 00:31:12.250 --> 00:31:14.870 I think with the lipids you can see them by eyeball, 00:31:14.870 --> 00:31:18.670 but you might look at absorption. 00:31:18.670 --> 00:31:23.560 If they have proteins, you could monitor absorption 00:31:23.560 --> 00:31:27.040 through the gradient, and that might tell you 00:31:27.040 --> 00:31:28.240 how to fractionate things. 00:31:28.240 --> 00:31:29.980 But anyhow, that's also an issue. 00:31:29.980 --> 00:31:33.790 Because before they can do the next step in the analysis, 00:31:33.790 --> 00:31:37.000 they've got to get the material out. 00:31:37.000 --> 00:31:40.660 So they've got the material out in each of these steps, 00:31:40.660 --> 00:31:42.930 and then, how do they look at this? 00:31:42.930 --> 00:31:45.970 They can pull it out. 00:31:45.970 --> 00:31:49.200 So what are they looking for? 00:31:49.200 --> 00:31:55.850 To tell them how effective this method is. 00:31:55.850 --> 00:31:59.230 AUDIENCE: Maybe some specific markers for each protein. 00:31:59.230 --> 00:32:00.280 JOANNE STUBBE: Exactly. 00:32:00.280 --> 00:32:03.640 So what are they-- 00:32:03.640 --> 00:32:05.860 to do that, what they're going to have to do 00:32:05.860 --> 00:32:10.450 is, before we look at the details of the method, 00:32:10.450 --> 00:32:12.760 I want to go through a western blot. 00:32:12.760 --> 00:32:15.087 So what do we know about a western blot? 00:32:15.087 --> 00:32:18.217 AUDIENCE: I have a quick question about the method here. 00:32:18.217 --> 00:32:19.800 JOANNE STUBBE: About the which method? 00:32:19.800 --> 00:32:21.383 AUDIENCE: The lysis method [INAUDIBLE] 00:32:21.383 --> 00:32:23.162 ball bearing homogenizer. 00:32:23.162 --> 00:32:26.230 So they're literally putting these cells in something 00:32:26.230 --> 00:32:27.481 like a bunch of ball bearings? 00:32:27.481 --> 00:32:29.105 JOANNE STUBBE: Yeah, you could do that. 00:32:29.105 --> 00:32:30.860 There's a lot of ways to crack open cells. 00:32:30.860 --> 00:32:32.410 I don't know which one's the best-- 00:32:32.410 --> 00:32:34.799 mammalian cells are really easy to open. 00:32:34.799 --> 00:32:36.340 Sometimes what I like to do is freeze 00:32:36.340 --> 00:32:37.756 and thaw them-- sometimes you have 00:32:37.756 --> 00:32:41.100 like a little mortar and pestle, or something like that. 00:32:41.100 --> 00:32:45.310 But that's-- I mean, yeast cells, you roll them. 00:32:45.310 --> 00:32:47.804 You have to have enough cells so you can do something. 00:32:47.804 --> 00:32:49.470 If you only have a tiny amount of cells, 00:32:49.470 --> 00:32:51.219 it makes it really challenging with beads, 00:32:51.219 --> 00:32:53.664 because it covers the beads. 00:32:53.664 --> 00:32:56.330 AUDIENCE: Do you have any issues with any of the different types 00:32:56.330 --> 00:32:57.177 of membranes that-- 00:32:57.177 --> 00:32:58.510 JOANNE STUBBE: Sticking to that? 00:32:58.510 --> 00:32:59.110 Absolutely. 00:32:59.110 --> 00:33:01.520 I'm sure you have to look at all of that kind of stuff. 00:33:01.520 --> 00:33:04.180 So how you choose, that's an important thing 00:33:04.180 --> 00:33:06.460 to look at, how you choose to crack open the cells. 00:33:06.460 --> 00:33:09.490 And it's the same with bacterial cells-- 00:33:09.490 --> 00:33:11.780 there are three or four ways to crack open the cells. 00:33:11.780 --> 00:33:15.490 And I can tell you only one of them really works efficiently. 00:33:15.490 --> 00:33:18.550 And a lot of people, when they use some of the others, 00:33:18.550 --> 00:33:20.500 they do something and they assume it works, 00:33:20.500 --> 00:33:22.720 but they never check to see whether the cell 00:33:22.720 --> 00:33:24.520 walls have been cracked open. 00:33:24.520 --> 00:33:26.950 A lot of times they haven't, and so what you get out 00:33:26.950 --> 00:33:30.040 is very, very low levels of protein, 00:33:30.040 --> 00:33:32.230 because you haven't cracked open the cell. 00:33:32.230 --> 00:33:34.960 So figuring out-- mammalian cells are apparently, 00:33:34.960 --> 00:33:36.430 I haven't worked with those myself, 00:33:36.430 --> 00:33:39.040 but they're apparently much easier 00:33:39.040 --> 00:33:41.200 to disrupt than bacteria. 00:33:41.200 --> 00:33:43.970 Or if you look at fungi-- 00:33:43.970 --> 00:33:46.630 fungi are really hard to crack open, yeast. 00:33:49.280 --> 00:33:52.180 So anyhow, that's an important thing to look at. 00:33:52.180 --> 00:33:57.340 So every one of these things, again, the devil 00:33:57.340 --> 00:33:58.284 is in the details. 00:33:58.284 --> 00:33:59.950 But when you're doing your own research, 00:33:59.950 --> 00:34:02.650 it doesn't matter what method you're looking at. 00:34:02.650 --> 00:34:05.860 The first time around, you need to look at it in detail, 00:34:05.860 --> 00:34:08.409 and convince yourself that this is a good way 00:34:08.409 --> 00:34:10.449 to chase this down. 00:34:10.449 --> 00:34:12.592 And you look at it in detail the first time around. 00:34:12.592 --> 00:34:14.050 And when you convince yourself it's 00:34:14.050 --> 00:34:16.175 working really well, and doing what you want to do, 00:34:16.175 --> 00:34:17.698 then you just use it. 00:34:17.698 --> 00:34:18.739 And that's the end of it. 00:34:18.739 --> 00:34:21.197 You don't have to go back and keep thinking about this over 00:34:21.197 --> 00:34:22.520 and over again. 00:34:22.520 --> 00:34:26.560 So the method we're going to use is a western blot. 00:34:26.560 --> 00:34:31.090 So we've got this stuff out, and have you all 00:34:31.090 --> 00:34:32.790 run SDS page shells? 00:34:32.790 --> 00:34:39.587 OK, so SDS page shells separate proteins how? 00:34:39.587 --> 00:34:40.670 AUDIENCE: Based on size... 00:34:40.670 --> 00:34:41.794 JOANNE STUBBE: By the what? 00:34:41.794 --> 00:34:46.760 AUDIENCE: It separates into a a charge gradient, and then-- 00:34:46.760 --> 00:34:48.967 not a charge gradient, but-- 00:34:48.967 --> 00:34:50.719 JOANNE STUBBE: Not charge. 00:34:50.719 --> 00:34:53.000 AUDIENCE: That's what drives the protein, but... 00:34:53.000 --> 00:34:55.449 JOANNE STUBBE: Right, but it's based on size, 00:34:55.449 --> 00:34:57.030 because it's coded-- 00:34:57.030 --> 00:35:01.580 every protein ratio is coded with this detergent, sodium 00:35:01.580 --> 00:35:04.750 dodecyl sulfate, which makes them migrate pretty 00:35:04.750 --> 00:35:06.860 much like the molecular weight. 00:35:06.860 --> 00:35:09.220 But if you've done these, it's not exactly 00:35:09.220 --> 00:35:10.360 like the molecular weight. 00:35:10.360 --> 00:35:13.030 You can do standards where you know the molecular weight, 00:35:13.030 --> 00:35:14.950 you can do a standard curve, and then 00:35:14.950 --> 00:35:17.590 you see where your protein migrates. 00:35:17.590 --> 00:35:20.140 And sometimes they migrate a little faster, sometimes 00:35:20.140 --> 00:35:22.900 a little slower, but it's OK. 00:35:22.900 --> 00:35:26.440 So you run this, and then what do you do? 00:35:26.440 --> 00:35:29.020 Does anybody know what you do next, to do a western? 00:35:31.530 --> 00:35:33.626 AUDIENCE: You need to use the membrane to... 00:35:33.626 --> 00:35:35.250 JOANNE STUBBE: Right, so the next thing 00:35:35.250 --> 00:35:37.820 they did was they used-- 00:35:37.820 --> 00:35:41.070 I'm going to put all of these up-- so they transferred it 00:35:41.070 --> 00:35:43.050 to a membrane. 00:35:43.050 --> 00:35:47.520 And why did they have to transfer it to a membrane 00:35:47.520 --> 00:35:50.460 to do this analysis? 00:35:50.460 --> 00:35:51.770 This is an extra step. 00:35:51.770 --> 00:35:52.570 And it turns out-- 00:35:56.480 --> 00:35:58.640 we're going to look at an antibody interacting 00:35:58.640 --> 00:35:59.300 with a protein. 00:35:59.300 --> 00:36:01.520 Why don't we just look at the antibody interacting 00:36:01.520 --> 00:36:03.393 with the protein to start with? 00:36:03.393 --> 00:36:04.490 AUDIENCE: It doesn't have access to the protein. 00:36:04.490 --> 00:36:05.890 JOANNE STUBBE: Right, it doesn't have very good access. 00:36:05.890 --> 00:36:07.830 It's really not very efficient. 00:36:07.830 --> 00:36:12.230 So people found, pretty much by trial and error, 00:36:12.230 --> 00:36:14.660 that you needed to transfer this to a membrane. 00:36:14.660 --> 00:36:16.910 I mean, we have hundreds of kinds of membranes. 00:36:16.910 --> 00:36:20.240 How did they choose nitrocellulose? 00:36:20.240 --> 00:36:22.510 If any of you have one run westerns, 00:36:22.510 --> 00:36:25.340 you remember what kind of a membrane you used? 00:36:25.340 --> 00:36:28.490 Did you use nitrocellulose? 00:36:28.490 --> 00:36:31.417 You do this in undergraduate class, don't you? 00:36:31.417 --> 00:36:32.375 You don't do a western? 00:36:35.320 --> 00:36:36.112 We used to do-- 00:36:36.112 --> 00:36:37.820 AUDIENCE: Did it once in undergrad class. 00:36:37.820 --> 00:36:40.310 JOANNE STUBBE: Yeah, in what kind of a membrane? 00:36:40.310 --> 00:36:41.180 Was it in biology? 00:36:41.180 --> 00:36:42.757 AUDIENCE: Yes, biology. 00:36:42.757 --> 00:36:44.090 JOANNE STUBBE: So what membrane? 00:36:44.090 --> 00:36:47.650 Do you remember what the membrane was? 00:36:47.650 --> 00:36:50.015 AUDIENCE: I think it was-- it was not nitrocellulose. 00:36:50.015 --> 00:36:51.640 JOANNE STUBBE: It's not nitrocellulose. 00:36:51.640 --> 00:36:56.860 So this PVDF, polyvinyl difluoride is the standard one 00:36:56.860 --> 00:36:57.767 that people use now. 00:36:57.767 --> 00:37:00.100 It works much better than nitrocellulose-- this paper is 00:37:00.100 --> 00:37:03.010 really old, and so they're looking at nitrocellulose. 00:37:03.010 --> 00:37:06.980 So then they do this. 00:37:06.980 --> 00:37:10.960 And then, what do they do next? 00:37:10.960 --> 00:37:12.550 They have an antibody-- 00:37:12.550 --> 00:37:14.740 we'll look at the details of this in a minute-- 00:37:14.740 --> 00:37:16.600 that can recognize the protein, that 00:37:16.600 --> 00:37:19.540 can find it on the membrane. 00:37:19.540 --> 00:37:21.520 And then what we're going to see is-- 00:37:21.520 --> 00:37:23.300 you still can't see anything really, 00:37:23.300 --> 00:37:25.620 because you don't have very much material there. 00:37:25.620 --> 00:37:27.100 And you can't observe-- 00:37:27.100 --> 00:37:29.640 you don't have enough to stain, oftentimes, by Coomassie, 00:37:29.640 --> 00:37:31.670 so you're going to have to amplify the signal. 00:37:31.670 --> 00:37:34.930 So then you're going to make an antibody to an antibody. 00:37:34.930 --> 00:37:36.710 And then you have to figure out how to, 00:37:36.710 --> 00:37:38.590 then, amplify the signal. 00:37:38.590 --> 00:37:41.170 And we'll look at that in a second. 00:37:41.170 --> 00:37:44.260 Is this what-- you ran a western, is 00:37:44.260 --> 00:37:45.990 this what westerns look like? 00:37:45.990 --> 00:37:48.490 AUDIENCE: I remember, we first [INAUDIBLE] 00:37:48.490 --> 00:37:50.710 non-specific proteins to occupy the sites. 00:37:50.710 --> 00:37:52.210 JOANNE STUBBE: Yeah, so that's good, 00:37:52.210 --> 00:37:55.540 you have to block everything, if you're using crude extract. 00:37:55.540 --> 00:38:00.610 So in this case, we would be using the crude mixture-- 00:38:00.610 --> 00:38:02.110 well, not a crude mixture, it's been 00:38:02.110 --> 00:38:05.641 fractured by the ultracentrifugation that's 00:38:05.641 --> 00:38:06.390 been fractionated. 00:38:06.390 --> 00:38:09.290 But you still have mixtures of proteins in there. 00:38:09.290 --> 00:38:14.240 Have any of you ever looked at westerns in a paper? 00:38:14.240 --> 00:38:18.460 Or even the papers you had to read? 00:38:18.460 --> 00:38:20.290 The paper on the PC-- 00:38:20.290 --> 00:38:23.460 go look at the PCK-- 00:38:23.460 --> 00:38:26.890 PCSK9 paper, that had westerns in it. 00:38:26.890 --> 00:38:27.630 What do you see? 00:38:27.630 --> 00:38:33.170 Do people show you something that looks like this? 00:38:33.170 --> 00:38:36.839 And if they did show you that, what would it look like? 00:38:36.839 --> 00:38:39.130 So you have an antibody that's specific for the protein 00:38:39.130 --> 00:38:42.814 of interest, whatever that is-- supposedly specific. 00:38:42.814 --> 00:38:43.480 What do you see? 00:38:47.220 --> 00:38:48.350 What do you think you see? 00:38:48.350 --> 00:38:49.891 Do you think antibodies are specific? 00:38:52.410 --> 00:38:54.740 I think I have an example of a typical western. 00:38:54.740 --> 00:38:57.877 AUDIENCE: I don't think they're as specific as [INAUDIBLE] 00:38:57.877 --> 00:38:58.710 JOANNE STUBBE: Yeah. 00:38:58.710 --> 00:38:59.210 Yeah. 00:38:59.210 --> 00:39:03.669 So when you look at a paper, you should pay attention 00:39:03.669 --> 00:39:05.960 to this when you read a paper, if you're doing anything 00:39:05.960 --> 00:39:07.126 in biology, what do you see? 00:39:07.126 --> 00:39:09.680 You never see a gel, ever. 00:39:09.680 --> 00:39:17.339 What you see is a slice of a gel where they cut off 00:39:17.339 --> 00:39:19.880 this-- the way they cut up all this stuff and all this stuff. 00:39:19.880 --> 00:39:23.000 The reason they do that is because it's a hell of a mess. 00:39:23.000 --> 00:39:25.610 So let me just show you a typical-- 00:39:25.610 --> 00:39:27.920 I don't care what kind of an antibody 00:39:27.920 --> 00:39:30.740 you're using, in crude extracts, it's a mess. 00:39:30.740 --> 00:39:33.710 Because you have non-specific interactions. 00:39:33.710 --> 00:39:36.045 We'll just look at that. 00:39:36.045 --> 00:39:38.420 So that would be something like you might see-- depending 00:39:38.420 --> 00:39:40.290 on how much antibody you have. 00:39:40.290 --> 00:39:43.220 So when you see this, the reason everybody 00:39:43.220 --> 00:39:45.220 reports data like that now. 00:39:45.220 --> 00:39:49.130 So it looks like it's really clean, but in reality-- 00:39:49.130 --> 00:39:53.040 I think if it is dirty as that, then in my opinion, 00:39:53.040 --> 00:39:55.550 I would make you publish the whole gel. 00:39:55.550 --> 00:39:56.660 But people don't do that. 00:39:56.660 --> 00:39:59.840 They just cut off the little band 00:39:59.840 --> 00:40:02.840 they're interested in-- they can see it change in concentration 00:40:02.840 --> 00:40:05.097 using this method. 00:40:05.097 --> 00:40:07.680 But you should be aware of the fact that antibodies in general 00:40:07.680 --> 00:40:09.960 aren't as specific as you think they're going to be. 00:40:09.960 --> 00:40:11.206 Yeah? 00:40:11.206 --> 00:40:14.767 AUDIENCE: Are they required to report the whole gel in 00:40:14.767 --> 00:40:15.350 supplementals? 00:40:15.350 --> 00:40:17.686 JOANNE STUBBE: I mean, I think, it probably 00:40:17.686 --> 00:40:19.310 depends on the journal, and it probably 00:40:19.310 --> 00:40:20.730 depends on the reviewer. 00:40:20.730 --> 00:40:23.990 But I would say, we're going away from data-- 00:40:23.990 --> 00:40:27.700 is something that is a pet peeve for me. 00:40:27.700 --> 00:40:30.110 And all the data, which I think is all right, 00:40:30.110 --> 00:40:32.390 is published in supplementary information, 00:40:32.390 --> 00:40:33.390 as opposed to the paper. 00:40:33.390 --> 00:40:35.480 I think if you have something really dirty, 00:40:35.480 --> 00:40:38.680 you should publish in the paper, in the main body of the paper. 00:40:38.680 --> 00:40:40.430 If you have something that's really clean, 00:40:40.430 --> 00:40:42.179 and it looks like that, it's fine with me. 00:40:42.179 --> 00:40:43.790 You don't even have to publish it, 00:40:43.790 --> 00:40:46.880 if you could believe what people were saying. 00:40:46.880 --> 00:40:49.370 Because people know what this looks like, 00:40:49.370 --> 00:40:51.380 a lot of people-- everybody uses westerns. 00:40:51.380 --> 00:40:53.690 But if it's a real mess, then you 00:40:53.690 --> 00:40:58.070 need to let your reader know that this is not 00:40:58.070 --> 00:41:00.380 such an easy experiment, and it's not so clear-cut. 00:41:00.380 --> 00:41:03.500 That's what your objective is, is to show people 00:41:03.500 --> 00:41:06.650 the data from which you drew your conclusions. 00:41:06.650 --> 00:41:08.600 And then they can draw their own conclusions, 00:41:08.600 --> 00:41:09.870 which may be different. 00:41:12.410 --> 00:41:15.080 So let's look at the apparatus to do this. 00:41:15.080 --> 00:41:19.590 So how do you get from here to here? 00:41:19.590 --> 00:41:22.880 So you have a gel, you run the gel, a polyacrylamide gel-- 00:41:22.880 --> 00:41:23.950 what do you do? 00:41:23.950 --> 00:41:25.610 AUDIENCE: Put the membrane on the gel. 00:41:25.610 --> 00:41:27.000 JOANNE STUBBE: So you put the membrane on the gel. 00:41:27.000 --> 00:41:27.791 And what do you do? 00:41:32.995 --> 00:41:35.030 AUDIENCE: [INAUDIBLE] applying charges to. 00:41:35.030 --> 00:41:38.210 JOANNE STUBBE: Yeah, so you're transferring it 00:41:38.210 --> 00:41:42.420 based on applying a voltage across this system. 00:41:42.420 --> 00:41:45.570 So here's your gel. 00:41:45.570 --> 00:41:51.980 And here's your membrane, nitrocellulose membrane. 00:41:51.980 --> 00:41:56.000 And then they have filter paper above the gel, 00:41:56.000 --> 00:41:57.230 and below the membrane. 00:41:57.230 --> 00:42:01.040 Why do you think they have the filter paper there? 00:42:01.040 --> 00:42:03.455 When you ran the gel, did you have filter paper? 00:42:03.455 --> 00:42:04.090 AUDIENCE: Yes. 00:42:04.090 --> 00:42:05.046 JOANNE STUBBE: Yeah. 00:42:09.830 --> 00:42:12.280 How do you think they decide how to do this transfer? 00:42:12.280 --> 00:42:16.220 Do you think is a straightforward? 00:42:16.220 --> 00:42:19.470 Do you run it for an hour, do you run it for five hours, 00:42:19.470 --> 00:42:22.020 do you run it for 15 minutes? 00:42:22.020 --> 00:42:24.440 What is the voltage you use to do the transfer? 00:42:24.440 --> 00:42:27.620 Do you think any of that is hard to figure out? 00:42:27.620 --> 00:42:29.930 So how do you figure that out? 00:42:29.930 --> 00:42:33.820 Somebody told you that this is a good way to do it? 00:42:33.820 --> 00:42:36.410 Yeah, so that might be a place you start. 00:42:36.410 --> 00:42:39.500 So you do it because somebody gave you a recipe. 00:42:39.500 --> 00:42:42.230 But then what do you need to do to make 00:42:42.230 --> 00:42:46.604 sure this recipe is correct? 00:42:46.604 --> 00:42:48.020 AUDIENCE: Find out what conditions 00:42:48.020 --> 00:42:50.060 that work for what you're working on. 00:42:50.060 --> 00:42:52.520 JOANNE STUBBE: Right, and then how do you do that? 00:42:52.520 --> 00:42:56.620 So that's true, every protein is going to be different. 00:42:56.620 --> 00:42:58.886 And if you have a protein-- 00:42:58.886 --> 00:43:03.800 if you have a clean protein, versus a mess of proteins, 00:43:03.800 --> 00:43:06.410 and you try to do this transfer, the transfer conditions 00:43:06.410 --> 00:43:08.070 will be different. 00:43:08.070 --> 00:43:10.220 So for example, if you really want 00:43:10.220 --> 00:43:13.480 to look at the concentration of something inside the cell, 00:43:13.480 --> 00:43:16.220 in the crude extracts, you never compare it 00:43:16.220 --> 00:43:18.710 to a standard with clean protein, 00:43:18.710 --> 00:43:21.790 because this transfer is different. 00:43:21.790 --> 00:43:23.720 So you need-- in the back of your mind, 00:43:23.720 --> 00:43:25.910 if you care about quantitating this, 00:43:25.910 --> 00:43:29.090 you need to understand the basis of the transfer. 00:43:29.090 --> 00:43:34.920 So why do you think they have these filter papers here? 00:43:34.920 --> 00:43:36.830 So this goes back to what controls 00:43:36.830 --> 00:43:42.890 you would do to see whether your transfer was working. 00:43:42.890 --> 00:43:44.120 So what would you look for? 00:43:49.630 --> 00:43:51.660 Did you do this? 00:43:51.660 --> 00:43:54.110 What did you do? 00:43:54.110 --> 00:43:56.716 What did you do with the filter papers in your-- 00:43:56.716 --> 00:44:01.526 AUDIENCE: You want to filter all to the SDS molecules... 00:44:01.526 --> 00:44:02.692 JOANNE STUBBE: You did what? 00:44:02.692 --> 00:44:04.110 AUDIENCE: You want to filter all-- 00:44:04.110 --> 00:44:05.960 JOANNE STUBBE: No, that's not what you do. 00:44:05.960 --> 00:44:07.710 I mean, you might want to do some of that, 00:44:07.710 --> 00:44:11.370 too, but in terms of thinking about whether your transfer is 00:44:11.370 --> 00:44:13.030 successful-- 00:44:13.030 --> 00:44:16.860 figuring out the conditions to blot from the gel 00:44:16.860 --> 00:44:20.740 to a piece of paper is not trivial. 00:44:20.740 --> 00:44:24.660 And there is a standard way that you do this, initially, to try. 00:44:24.660 --> 00:44:27.180 But then you have to make sure that that method is working. 00:44:27.180 --> 00:44:28.746 And lots of times it doesn't work. 00:44:28.746 --> 00:44:30.870 So it's something that's going to be experimentally 00:44:30.870 --> 00:44:32.340 determined. 00:44:32.340 --> 00:44:35.640 So the question is, what would you 00:44:35.640 --> 00:44:38.880 think would happen if you did this for six or seven hours? 00:44:38.880 --> 00:44:43.450 Whereas, a normal blot would take two hours? 00:44:43.450 --> 00:44:45.730 AUDIENCE: Would be transferred onto the filter paper? 00:44:45.730 --> 00:44:47.760 JOANNE STUBBE: Right, it would go right into the filter paper, 00:44:47.760 --> 00:44:49.150 or even off the filter paper. 00:44:49.150 --> 00:44:51.150 So what you do is you take the filter paper out, 00:44:51.150 --> 00:44:54.360 you look for protein being bound. 00:44:54.360 --> 00:44:56.880 What about the gel? 00:44:56.880 --> 00:45:01.016 What do you do with the gel after your experiment's over? 00:45:01.016 --> 00:45:03.457 AUDIENCE: Make sure a protein's not on it? 00:45:03.457 --> 00:45:06.040 JOANNE STUBBE: Right, make sure that the protein is not on it. 00:45:06.040 --> 00:45:07.780 So these are simple controls, but these 00:45:07.780 --> 00:45:10.180 are the controls you always do until you work out 00:45:10.180 --> 00:45:12.590 the conditions to make sure this works. 00:45:12.590 --> 00:45:16.390 And it's pretty critical to make sure you have good transfer. 00:45:16.390 --> 00:45:21.730 So then, so this is the antibody thing that they do. 00:45:21.730 --> 00:45:23.920 Has anybody thought about these kinds of assays? 00:45:23.920 --> 00:45:26.170 You've seen them, I think, already in class. 00:45:26.170 --> 00:45:28.677 But what's wrong with this picture? 00:45:28.677 --> 00:45:30.760 The target protein, what's wrong with this picture 00:45:30.760 --> 00:45:31.400 in the target? 00:45:31.400 --> 00:45:34.300 So here's your nitrocellulose filter paper. 00:45:34.300 --> 00:45:36.040 What's wrong with this cartoon? 00:45:39.950 --> 00:45:41.400 Should be unfolded, yeah. 00:45:41.400 --> 00:45:44.130 So you're doing SDS page, it's unfolded. 00:45:44.130 --> 00:45:46.260 So then we react it with an antibody. 00:45:46.260 --> 00:45:48.210 Presumably we have a good antibody, 00:45:48.210 --> 00:45:50.490 but you've already learned in the first half 00:45:50.490 --> 00:45:53.520 of this course that having really good antibodies is not 00:45:53.520 --> 00:45:54.270 so trivial-- 00:45:54.270 --> 00:45:57.750 you can get them, but most of the time 00:45:57.750 --> 00:46:01.680 they are not specific if you're looking at crude extracts. 00:46:01.680 --> 00:46:03.630 They have little epitopes they recognize, 00:46:03.630 --> 00:46:05.490 if you're using monoclonals that could 00:46:05.490 --> 00:46:08.860 be present in other proteins. 00:46:08.860 --> 00:46:14.730 And furthermore, how are you detecting something? 00:46:14.730 --> 00:46:17.910 An antibody as a protein, it has absorption of 280. 00:46:17.910 --> 00:46:22.140 Again, this is too low to see, so putting an antibody on it 00:46:22.140 --> 00:46:24.216 is still going to be too low to detect. 00:46:24.216 --> 00:46:25.590 So how do you detect your signal? 00:46:28.440 --> 00:46:30.130 So have you done this? 00:46:30.130 --> 00:46:32.830 I'm surprised they don't do this in your introductory class-- 00:46:32.830 --> 00:46:35.380 they don't do westerns, at all. 00:46:35.380 --> 00:46:40.210 So what you're looking at is an antibody to an antibody. 00:46:40.210 --> 00:46:41.770 So you put your antibody on, that's 00:46:41.770 --> 00:46:43.280 specific for your protein. 00:46:43.280 --> 00:46:46.150 And then you make an antibody in another organism 00:46:46.150 --> 00:46:51.040 that can specifically recognize antibodies in general. 00:46:51.040 --> 00:46:55.940 So if this is to a mouse, you make it to go and isolate that. 00:46:55.940 --> 00:47:00.070 And then what you do is derivatize the second antibody 00:47:00.070 --> 00:47:00.700 with what? 00:47:00.700 --> 00:47:02.740 A protein? 00:47:02.740 --> 00:47:06.322 That can function as a catalyst. 00:47:06.322 --> 00:47:08.780 AUDIENCE: Why can't you just derivatize the first antibody? 00:47:08.780 --> 00:47:11.752 JOANNE STUBBE: Well, what? 00:47:11.752 --> 00:47:12.460 What did you say? 00:47:12.460 --> 00:47:13.930 AUDIENCE: It's more expensive? 00:47:13.930 --> 00:47:16.013 JOANNE STUBBE: Well, no, I don't know whether it's 00:47:16.013 --> 00:47:17.760 more expensive or not. 00:47:17.760 --> 00:47:19.062 But-- 00:47:19.062 --> 00:47:21.330 AUDIENCE: Well, because you'd have to derivatize 00:47:21.330 --> 00:47:22.290 every primary antibody. 00:47:22.290 --> 00:47:24.331 JOANNE STUBBE: So you'd have the derivatize every 00:47:24.331 --> 00:47:27.100 primary antibody, and so this is a standard procedure. 00:47:27.100 --> 00:47:30.080 You could derivatize the primary antibody. 00:47:30.080 --> 00:47:32.716 So that's not a bad question. 00:47:32.716 --> 00:47:34.090 And so what you're doing now, you 00:47:34.090 --> 00:47:38.890 can buy these commercially, so they have rabbit, rabbit, 00:47:38.890 --> 00:47:40.930 mouse, whatever, antibodies. 00:47:40.930 --> 00:47:44.230 And the key is the amplification of the signal, 00:47:44.230 --> 00:47:48.370 and you use enzymes to amplify the signal. 00:47:48.370 --> 00:47:50.620 Does anybody know what the enzymes 00:47:50.620 --> 00:47:54.348 are, what the enzymes do to amplify the signal? 00:47:54.348 --> 00:48:00.569 AUDIENCE: You can covert the molecule to a blue molecule... 00:48:00.569 --> 00:48:02.360 JOANNE STUBBE: To something that's colored. 00:48:02.360 --> 00:48:05.780 So does anybody know what that horseradish peroxidase-- 00:48:05.780 --> 00:48:08.360 have you ever heard of horseradish peroxidase? 00:48:08.360 --> 00:48:10.750 So that's a heme iron-- we're going 00:48:10.750 --> 00:48:12.650 to be talking about heme irons pretty soon, 00:48:12.650 --> 00:48:14.210 and hydrogen peroxide. 00:48:14.210 --> 00:48:17.000 It makes a chemically very reactive iron oxide 00:48:17.000 --> 00:48:22.190 species, that can oxidize a dye that changes color. 00:48:22.190 --> 00:48:24.300 And it has extremely high extinction coefficients. 00:48:24.300 --> 00:48:26.819 So you can see it, and it does it catalytically 00:48:26.819 --> 00:48:28.610 and the lifetime of the dye is long enough. 00:48:28.610 --> 00:48:30.650 So it accumulates, and you can get really 00:48:30.650 --> 00:48:32.030 amplification of your signal. 00:48:32.030 --> 00:48:35.780 Or you can use a phosphatase that 00:48:35.780 --> 00:48:38.840 liberates something that's highly colored, again, 00:48:38.840 --> 00:48:39.870 and you can see it. 00:48:39.870 --> 00:48:45.180 So this is a standard method that everybody uses. 00:48:45.180 --> 00:48:48.050 And so, that's our gel. 00:48:48.050 --> 00:48:51.380 So now we're looking at sort of-- 00:48:51.380 --> 00:48:53.990 at the end already-- but we're looking at these gels, 00:48:53.990 --> 00:48:56.580 and what do you see through the different steps? 00:48:56.580 --> 00:49:00.830 So if we look through the first gradient, 00:49:00.830 --> 00:49:05.890 through the sucrose gradient, that gets us through DNE. 00:49:05.890 --> 00:49:10.580 And if you look, say, at lane E-- 00:49:10.580 --> 00:49:14.990 our goal is to separate proteins that 00:49:14.990 --> 00:49:18.450 are specifically localized in each one of these membranes. 00:49:18.450 --> 00:49:20.810 So you need to believe that's true, 00:49:20.810 --> 00:49:24.470 that people have selected the right group of proteins 00:49:24.470 --> 00:49:25.120 to look for. 00:49:25.120 --> 00:49:27.974 And you notice they do more than one. 00:49:27.974 --> 00:49:29.390 So they look at multiple proteins. 00:49:29.390 --> 00:49:31.090 Why do you think-- 00:49:31.090 --> 00:49:35.770 do you think it's easy to select the proteins to look for? 00:49:35.770 --> 00:49:38.220 And why or why not? 00:49:38.220 --> 00:49:41.240 So, they obviously have selected a group of proteins, 00:49:41.240 --> 00:49:44.240 and I think most people would agree that they've selected 00:49:44.240 --> 00:49:46.160 a good group of proteins. 00:49:46.160 --> 00:49:48.980 But what do we know now about proteins, 00:49:48.980 --> 00:49:51.706 do they stay in one place? 00:49:51.706 --> 00:49:55.760 No, they move around. 00:49:55.760 --> 00:49:58.250 But some might be present in very low amounts, 00:49:58.250 --> 00:49:59.960 sometimes in much higher amounts. 00:49:59.960 --> 00:50:04.340 And so you need to have more than one protein as a control 00:50:04.340 --> 00:50:06.800 to make sure you're looking in the right region. 00:50:06.800 --> 00:50:09.500 And what do you see in E? 00:50:09.500 --> 00:50:12.900 If you look over here, it tells you what the organelle is. 00:50:12.900 --> 00:50:16.400 And if you look at this protein, this 00:50:16.400 --> 00:50:19.880 is localized to the lisosomes-- we talked about that in class. 00:50:19.880 --> 00:50:22.010 If you looked at this protein, it's 00:50:22.010 --> 00:50:24.080 localized to the peroxisomes. 00:50:24.080 --> 00:50:29.360 So in addition to the ones we care about, the ER proteins, 00:50:29.360 --> 00:50:31.730 we're also getting proteins that are 00:50:31.730 --> 00:50:34.010 localized in other membranes. 00:50:34.010 --> 00:50:39.320 So that's when they went to the next method, 00:50:39.320 --> 00:50:42.200 and they added on another gradient 00:50:42.200 --> 00:50:46.290 to try to separate out, again, the lysosomal 00:50:46.290 --> 00:50:49.900 and the peroxisomal proteins. 00:50:49.900 --> 00:50:52.740 And you can see they were pretty successful at this. 00:50:52.740 --> 00:50:56.180 There's none of these proteins left in this gradient. 00:50:56.180 --> 00:50:58.370 So that's good. 00:50:58.370 --> 00:50:59.790 And they took it a step further. 00:50:59.790 --> 00:51:01.250 Do you remember what this is? 00:51:01.250 --> 00:51:04.202 What are they looking for down here, in this? 00:51:04.202 --> 00:51:05.410 AUDIENCE: Enzymatic activity. 00:51:05.410 --> 00:51:07.160 JOANNE STUBBE: Yeah, so enzymatic activity 00:51:07.160 --> 00:51:08.840 is localized in certain organelles. 00:51:08.840 --> 00:51:11.090 So they again did a second experiment 00:51:11.090 --> 00:51:12.720 to look at all of that. 00:51:12.720 --> 00:51:14.950 So they were very careful in this, 00:51:14.950 --> 00:51:16.340 they figured out how to separate. 00:51:16.340 --> 00:51:17.780 And that's the key thing for them 00:51:17.780 --> 00:51:22.220 to analyzing the concentration of cholesterol 00:51:22.220 --> 00:51:23.180 in these membranes. 00:51:23.180 --> 00:51:25.130 And what they looked at-- we're over time-- 00:51:25.130 --> 00:51:27.980 but is the concentration of cholesterol compared 00:51:27.980 --> 00:51:30.570 to the total amount of lipids. 00:51:30.570 --> 00:51:34.220 And how did they do that analysis? 00:51:34.220 --> 00:51:36.860 Gene Kennedy, who's at Harvard Medical School-- 00:51:36.860 --> 00:51:39.530 he's in his 90s, now-- really trained all the lipid 00:51:39.530 --> 00:51:42.250 chemists in the whole country. 00:51:42.250 --> 00:51:44.000 And they figured out many years ago 00:51:44.000 --> 00:51:47.187 how to separate lipid fractions with methanol, chloroform 00:51:47.187 --> 00:51:49.520 extract, something that you guys probably haven't though 00:51:49.520 --> 00:51:51.140 about at all. 00:51:51.140 --> 00:51:53.870 But we're really pretty good at separating things, 00:51:53.870 --> 00:51:56.090 and it's nothing more than an extraction 00:51:56.090 --> 00:52:00.410 like you do as organic chemist to purify and separate things. 00:52:00.410 --> 00:52:01.740 We've figured that out. 00:52:01.740 --> 00:52:07.220 And so then they use mass spec to allow them to quantitate 00:52:07.220 --> 00:52:09.680 the amount of glycerol. 00:52:09.680 --> 00:52:16.730 And then in the end, so they use mass spec, these western blots, 00:52:16.730 --> 00:52:20.510 and they can change the concentration 00:52:20.510 --> 00:52:23.420 of the cholesterol and do the experiments over and over 00:52:23.420 --> 00:52:26.660 again, to see what happens. 00:52:26.660 --> 00:52:32.180 And when they do that, this is the picture of cyclodextrin. 00:52:32.180 --> 00:52:35.060 So you can see the only difference is this group here 00:52:35.060 --> 00:52:38.020 versus that with a methyl. 00:52:38.020 --> 00:52:42.050 And one, so this is hydroxypropryl-- 00:52:42.050 --> 00:52:47.000 hydroxypropionyl cyclodextrin-- so 00:52:47.000 --> 00:52:50.240 it's like a cavity like this. 00:52:50.240 --> 00:52:53.330 And the other only other change here is a methyl group, 00:52:53.330 --> 00:52:54.500 removing that. 00:52:54.500 --> 00:52:57.230 And they have very different properties 00:52:57.230 --> 00:53:00.560 about binding and releasing cholesterol, which somebody 00:53:00.560 --> 00:53:04.310 had to do a lot of studying on to be able to ensure that they 00:53:04.310 --> 00:53:07.520 can use it to remove cholesterol, 00:53:07.520 --> 00:53:09.860 and then to add it back to the media. 00:53:09.860 --> 00:53:12.154 And so you have to think about the exchange kinetics, 00:53:12.154 --> 00:53:13.820 you have to think about a lot of things. 00:53:13.820 --> 00:53:16.760 This is not trivial to set this up, 00:53:16.760 --> 00:53:21.070 to figure out how to control the levels of cholesterol. 00:53:21.070 --> 00:53:24.560 And then what they do is, this is like a typical assay, 00:53:24.560 --> 00:53:26.480 and this is the end. 00:53:26.480 --> 00:53:29.960 What you can do is this, removes cholesterol, 00:53:29.960 --> 00:53:32.300 and you can see it change. 00:53:32.300 --> 00:53:37.940 This reports on low levels of cholesterol, 00:53:37.940 --> 00:53:41.090 which is happening over here, allows the protein 00:53:41.090 --> 00:53:43.820 to move to the nucleus where it's smaller. 00:53:43.820 --> 00:53:46.460 And that's how they do the correlation-- the correlation 00:53:46.460 --> 00:53:48.110 between the levels in the nucleus 00:53:48.110 --> 00:53:50.850 and the levels of cholesterol. 00:53:50.850 --> 00:53:53.960 So I thought this was a pretty cool paper. 00:53:53.960 --> 00:53:56.630 And these kinds of methods, I think, 00:53:56.630 --> 00:53:59.370 will be applicable to a wide range of things 00:53:59.370 --> 00:54:03.800 if people ever do biochemistry, looking 00:54:03.800 --> 00:54:05.470 at the function of membranes. 00:54:05.470 --> 00:54:08.620 So, OK, guys.