1 00:00:00,960 --> 00:00:03,270 The following content is provided under a Creative 2 00:00:03,270 --> 00:00:04,630 Commons license. 3 00:00:04,630 --> 00:00:07,140 Your support will help MIT OpenCourseWare 4 00:00:07,140 --> 00:00:11,470 continue to offer high-quality educational resources for free. 5 00:00:11,470 --> 00:00:14,100 To make a donation, or view additional materials 6 00:00:14,100 --> 00:00:18,050 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:18,050 --> 00:00:19,000 at ocw.mit.edu. 8 00:00:24,807 --> 00:00:26,640 JOANNE STUBBE: This is the second recitation 9 00:00:26,640 --> 00:00:29,250 on cholesterol, and it's really focused 10 00:00:29,250 --> 00:00:34,680 on this question of how do you sense cholesterol 11 00:00:34,680 --> 00:00:36,030 in a membrane? 12 00:00:36,030 --> 00:00:39,540 So that's really a tough problem. 13 00:00:39,540 --> 00:00:43,020 And they've developed new tools, and that's 14 00:00:43,020 --> 00:00:45,470 what we're going to be talking about-- what the tools are, 15 00:00:45,470 --> 00:00:48,390 and whether you would think they were 16 00:00:48,390 --> 00:00:53,400 adequate to be able to address this question about what kinds 17 00:00:53,400 --> 00:00:56,380 of changes in concentration of cholesterol. 18 00:00:56,380 --> 00:00:58,000 Number one, can you measure them? 19 00:00:58,000 --> 00:01:01,620 And number two, what effects do they have, 20 00:01:01,620 --> 00:01:05,220 in terms of whether you're going to turn on cholesterol 21 00:01:05,220 --> 00:01:08,760 biosynthesis and uptake, because you need more cholesterol, 22 00:01:08,760 --> 00:01:11,490 or you're going to turn the whole thing off? 23 00:01:11,490 --> 00:01:16,110 So we've been focusing, as we've described 24 00:01:16,110 --> 00:01:20,010 in the last few lectures, in the endoplasmic reticulum. 25 00:01:20,010 --> 00:01:23,400 And what would the cholesterol-- 26 00:01:23,400 --> 00:01:25,920 what kinds of changes in cholesterols 27 00:01:25,920 --> 00:01:28,890 did they see in the experiments they were doing in this paper? 28 00:01:28,890 --> 00:01:34,709 What were the range of changes that they saw? 29 00:01:34,709 --> 00:01:36,010 AUDIENCE: 3% to 10%? 30 00:01:36,010 --> 00:01:39,300 JOANNE STUBBE: Yeah, so see, something low. 31 00:01:39,300 --> 00:01:42,840 Say they were trying to do this same experiment in the plasma 32 00:01:42,840 --> 00:01:45,450 membrane-- how do we know it's the ER membrane that 33 00:01:45,450 --> 00:01:46,775 does this sensing? 34 00:01:46,775 --> 00:01:48,850 That's what the whole paper is focused on, 35 00:01:48,850 --> 00:01:51,960 that's what everything we've focused on in class. 36 00:01:51,960 --> 00:01:54,870 Say you wanted to do a similar kind of experiment 37 00:01:54,870 --> 00:01:58,080 in the plasma membrane, do you remember 38 00:01:58,080 --> 00:02:01,460 what I said about the levels of cholesterol? 39 00:02:01,460 --> 00:02:04,650 So they distributed throughout the cell, in all membranes. 40 00:02:04,650 --> 00:02:06,330 Where is the most cholesterol? 41 00:02:09,490 --> 00:02:12,360 So if you don't remember, it's the plasma membrane. 42 00:02:12,360 --> 00:02:17,490 So say, instead of having 7% or 8% of the lipids cholesterol, 43 00:02:17,490 --> 00:02:20,460 say you had 40%-- 44 00:02:20,460 --> 00:02:22,410 that's an over-exaggeration-- do you 45 00:02:22,410 --> 00:02:26,360 think this kind of an experiment would be hard to do, 46 00:02:26,360 --> 00:02:30,380 that they've talked about in this paper? 47 00:02:30,380 --> 00:02:32,190 So you would want to do this-- if you 48 00:02:32,190 --> 00:02:34,785 tried to do the same experiment with the plasma membrane? 49 00:02:43,444 --> 00:02:45,360 So the key issue that you need to think about, 50 00:02:45,360 --> 00:02:48,030 is go back and look at the changes-- 51 00:02:48,030 --> 00:02:50,950 they did a whole bunch of different experiments. 52 00:02:50,950 --> 00:02:53,070 The numbers are squishy, but they came up 53 00:02:53,070 --> 00:02:56,250 with numbers that reproduced themselves, I thought, 54 00:02:56,250 --> 00:02:58,080 in an amazing way. 55 00:02:58,080 --> 00:02:59,990 But now say you wanted to do this 56 00:02:59,990 --> 00:03:06,090 in the plasma membrane, where the levels of cholesterol 57 00:03:06,090 --> 00:03:06,780 are much higher. 58 00:03:11,280 --> 00:03:13,150 Do you think it would be easy to do? 59 00:03:13,150 --> 00:03:15,440 Using the same tech techniques that 60 00:03:15,440 --> 00:03:19,470 are described, that we're going to discuss, or not? 61 00:03:19,470 --> 00:03:22,640 And what would the issues be? 62 00:03:22,640 --> 00:03:23,380 Yeah? 63 00:03:23,380 --> 00:03:25,426 AUDIENCE: So they had to deplete the cholesterol 64 00:03:25,426 --> 00:03:28,640 from the membrane, and so that would probably 65 00:03:28,640 --> 00:03:32,545 be hard to deplete it to a level that's low enough, so that you 66 00:03:32,545 --> 00:03:34,150 don't get the activity. 67 00:03:34,150 --> 00:03:34,651 Right? 68 00:03:34,651 --> 00:03:35,983 JOANNE STUBBE: So, I don't know. 69 00:03:35,983 --> 00:03:37,560 So that's an interesting question. 70 00:03:37,560 --> 00:03:38,870 So you'd have to deplete-- 71 00:03:38,870 --> 00:03:40,411 so that's going to be it, we're going 72 00:03:40,411 --> 00:03:42,710 to have to control the cholesterol levels. 73 00:03:42,710 --> 00:03:44,900 But what change-- if you looked at the changes 74 00:03:44,900 --> 00:03:48,665 in levels of cholesterol in the ER, how much did they change? 75 00:03:56,280 --> 00:03:57,760 They change from what to what? 76 00:03:57,760 --> 00:04:03,820 From-- 2% to 7%. 77 00:04:03,820 --> 00:04:07,120 Say that you were in that same range of change that 78 00:04:07,120 --> 00:04:12,370 was going to turn on a switch in the plasma membrane. 79 00:04:12,370 --> 00:04:14,500 And say you could control the levels. 80 00:04:14,500 --> 00:04:18,550 Do you think it would be easy to see that? 81 00:04:18,550 --> 00:04:23,762 So you start with 40%, say, that's the norm. 82 00:04:23,762 --> 00:04:26,910 Say the change was very similar to what 83 00:04:26,910 --> 00:04:28,760 you see in the change in the ER-- 84 00:04:28,760 --> 00:04:30,510 do you think that would be easy to detect? 85 00:04:30,510 --> 00:04:33,660 No, because now you have two big numbers, 86 00:04:33,660 --> 00:04:36,140 and there's a huge amount of error 87 00:04:36,140 --> 00:04:37,530 in this method of analysis. 88 00:04:37,530 --> 00:04:39,321 So those are the kinds of things I'm trying 89 00:04:39,321 --> 00:04:41,350 to get you to think about. 90 00:04:41,350 --> 00:04:43,800 I don't know why it's the ER-- 91 00:04:43,800 --> 00:04:46,470 I mean, everybody's focused on the ER. 92 00:04:46,470 --> 00:04:50,160 Could cholesterol and other organelles 93 00:04:50,160 --> 00:04:52,200 have a different regulatory mechanism? 94 00:04:52,200 --> 00:04:55,650 Or somehow be connected, still, to what's going on in the ER? 95 00:04:55,650 --> 00:04:59,880 Could be-- I mean, you start out with the simplest model 96 00:04:59,880 --> 00:05:03,450 you can get and you test it, but then as you learn more, 97 00:05:03,450 --> 00:05:05,910 or we have more and more technology, 98 00:05:05,910 --> 00:05:08,880 we learn new things, you go back and you revisit and rethink 99 00:05:08,880 --> 00:05:11,890 about what's going on. 100 00:05:11,890 --> 00:05:13,950 So the key question is, it's really 101 00:05:13,950 --> 00:05:17,430 this switch of having cholesterol 102 00:05:17,430 --> 00:05:20,866 that keeps it in the membrane, or not having cholesterol. 103 00:05:20,866 --> 00:05:22,740 And the question is, what are the differences 104 00:05:22,740 --> 00:05:28,740 in the levels that allow turn on of cholesterol-- biosynthesis 105 00:05:28,740 --> 00:05:34,950 and LDL biosynthesis, which then allows uptake of cholesterol 106 00:05:34,950 --> 00:05:35,790 from the diet? 107 00:05:35,790 --> 00:05:38,422 OK, so that's the question. 108 00:05:38,422 --> 00:05:39,630 And what does this look like? 109 00:05:39,630 --> 00:05:42,080 And people hadn't measured this by any method, 110 00:05:42,080 --> 00:05:45,480 and this model I've gone through a number of times in class 111 00:05:45,480 --> 00:05:47,520 today, so I'm not going to go through it again. 112 00:05:47,520 --> 00:05:49,830 Hopefully you all know that in some form in your head, 113 00:05:49,830 --> 00:05:53,870 or you have the picture in front of you so you can remember it. 114 00:05:53,870 --> 00:05:55,620 So these are the questions I want to pose, 115 00:05:55,620 --> 00:05:58,470 and I want you guys to do the talking today. 116 00:05:58,470 --> 00:06:02,980 And what I'm going to do is, I have most of the figures 117 00:06:02,980 --> 00:06:06,380 on my PowerPoint, so we can bring them up and look at them. 118 00:06:06,380 --> 00:06:08,110 And you can tell me what you see. 119 00:06:08,110 --> 00:06:13,070 And then everybody might be seeing something different-- 120 00:06:13,070 --> 00:06:16,380 and so we're thinking about this differently, 121 00:06:16,380 --> 00:06:20,190 and maybe we come to some kind of consensus about 122 00:06:20,190 --> 00:06:23,200 whether these experiments were carried out well or not. 123 00:06:23,200 --> 00:06:25,920 So one of the first things-- so these 124 00:06:25,920 --> 00:06:28,470 will be the general things, and then we'll step through them. 125 00:06:28,470 --> 00:06:33,150 But they wanted to perturb the cellular cholesterol levels. 126 00:06:33,150 --> 00:06:36,620 And how did they end up doing that? 127 00:06:36,620 --> 00:06:38,190 Did that make sense? 128 00:06:38,190 --> 00:06:40,360 We talked a little bit about this already. 129 00:06:40,360 --> 00:06:42,720 I mean, what did they use as tools to do that? 130 00:06:45,648 --> 00:06:47,469 AUDIENCE: [INAUDIBLE] 131 00:06:47,469 --> 00:06:49,260 JOANNE STUBBE: So you need to speak louder, 132 00:06:49,260 --> 00:06:50,301 because I really am deaf. 133 00:06:50,301 --> 00:06:52,230 Sorry. 134 00:06:52,230 --> 00:06:53,744 AUDIENCE: So just, right here, they 135 00:06:53,744 --> 00:06:56,160 were careful of the amount of cholesterol present in this? 136 00:06:56,160 --> 00:06:57,618 JOANNE STUBBE: So that's one place, 137 00:06:57,618 --> 00:06:59,550 so they can deplete cholesterol for the media. 138 00:06:59,550 --> 00:07:02,670 But then what did they do? 139 00:07:02,670 --> 00:07:04,800 So the whole paper is about this-- how did they 140 00:07:04,800 --> 00:07:06,030 control the [INAUDIBLE]? 141 00:07:06,030 --> 00:07:08,490 Let's assume that they can do that, 142 00:07:08,490 --> 00:07:09,820 and they got good at that. 143 00:07:09,820 --> 00:07:11,760 I think a lot of people have used that method, 144 00:07:11,760 --> 00:07:15,450 and so they can deplete media. 145 00:07:15,450 --> 00:07:20,340 So how did they deplete cholesterol? 146 00:07:20,340 --> 00:07:23,460 There was some unusual ways to deplete cholesterol 147 00:07:23,460 --> 00:07:24,690 in this paper. 148 00:07:24,690 --> 00:07:26,746 Did any of you pick up on that? 149 00:07:26,746 --> 00:07:29,520 AUDIENCE: A chemical that could bind to cholesterol. 150 00:07:29,520 --> 00:07:32,100 JOANNE STUBBE: So did you think that was unusual? 151 00:07:32,100 --> 00:07:34,150 Did any of you look up what that was? 152 00:07:34,150 --> 00:07:37,695 AUDIENCE: It was a kind of carbohydrate 153 00:07:37,695 --> 00:07:40,056 that can bind to cholesterol. 154 00:07:40,056 --> 00:07:42,560 JOANNE STUBBE: Yeah, so but what was interesting about it, 155 00:07:42,560 --> 00:07:45,890 it was hydroxypropyl-- 156 00:07:45,890 --> 00:07:48,570 remember HP, cyclodextrin. 157 00:07:48,570 --> 00:07:50,310 We're going to look at this in a minute. 158 00:07:50,310 --> 00:07:52,110 But what do we know-- 159 00:07:52,110 --> 00:07:57,380 what was the other molecule they used to add cholesterol back? 160 00:07:57,380 --> 00:08:01,110 AUDIENCE: Another form of that molecule is-- 161 00:08:01,110 --> 00:08:03,871 JOANNE STUBBE: So methyl-cyclodextrin-- 162 00:08:03,871 --> 00:08:05,370 I'm going to show you the structure, 163 00:08:05,370 --> 00:08:07,450 but they aren't very different. 164 00:08:07,450 --> 00:08:12,750 So have any of you ever heard of cyclodextrin before? 165 00:08:12,750 --> 00:08:16,170 People won the Nobel Prize for that, Don Cram won it, 166 00:08:16,170 --> 00:08:19,219 Breslow spent his whole life studying host guest 167 00:08:19,219 --> 00:08:19,760 interactions. 168 00:08:19,760 --> 00:08:23,700 So you guys, I don't know what you teach you now anymore, 169 00:08:23,700 --> 00:08:26,400 but that used to be something that was taught a lot, 170 00:08:26,400 --> 00:08:28,320 host guest interactions, trying to understand 171 00:08:28,320 --> 00:08:31,875 weak non-covalent interactions as the basis for understanding 172 00:08:31,875 --> 00:08:33,150 catalysis. 173 00:08:33,150 --> 00:08:34,590 But to me, that was-- 174 00:08:34,590 --> 00:08:36,990 immediately when I saw this, what the heck's going on? 175 00:08:36,990 --> 00:08:39,740 So then I Googled it, and immediately-- 176 00:08:39,740 --> 00:08:42,780 and I don't know anything about hydroxypropyl-- you Google it, 177 00:08:42,780 --> 00:08:43,919 you look it up. 178 00:08:43,919 --> 00:08:47,040 And then you look at it, and if you were a chemist 179 00:08:47,040 --> 00:08:49,980 and you were really interested in the molecular interactions, 180 00:08:49,980 --> 00:08:52,000 you might make a model of it. 181 00:08:52,000 --> 00:08:55,860 And then see, what is the difference between that one 182 00:08:55,860 --> 00:08:58,620 little group, when you look at the structure, it's amazing. 183 00:08:58,620 --> 00:09:02,670 And that's the basis of most of the experiments. 184 00:09:02,670 --> 00:09:06,240 So you need to believe that they figured that out. 185 00:09:06,240 --> 00:09:10,570 And that's not in this paper, so if you really cared about it 186 00:09:10,570 --> 00:09:12,720 you would have to go back and read earlier papers, 187 00:09:12,720 --> 00:09:16,470 and see what are the experiments that led them 188 00:09:16,470 --> 00:09:19,050 to focus on these molecules? 189 00:09:19,050 --> 00:09:23,430 How else did they end up getting cholesterol levels back 190 00:09:23,430 --> 00:09:24,060 into the cell? 191 00:09:24,060 --> 00:09:26,684 Do you remember what the other method was? 192 00:09:26,684 --> 00:09:29,100 So we'll come back and we'll talk about this in a minute-- 193 00:09:29,100 --> 00:09:30,391 so that was one of the methods. 194 00:09:35,760 --> 00:09:37,767 AUDIENCE: They added two kind of sterols. 195 00:09:37,767 --> 00:09:40,100 JOANNE STUBBE: OK, so they did add two kind of sterols-- 196 00:09:40,100 --> 00:09:41,516 and they tried to figure out, this 197 00:09:41,516 --> 00:09:45,200 is another unknown, what was the difference between the sterols? 198 00:09:45,200 --> 00:09:47,030 Simply a hydroxyl group. 199 00:09:47,030 --> 00:09:53,020 OK, so if you looked at this, cholesterol is this guy. 200 00:09:53,020 --> 00:09:57,080 And then they had something like this guy-- 201 00:09:57,080 --> 00:09:59,750 25, and remember where [INAUDIBLE] the side chain, 202 00:09:59,750 --> 00:10:06,080 hanging out of the little [? cheer ?] system you have. 203 00:10:06,080 --> 00:10:08,100 I don't think they learned very much from that. 204 00:10:08,100 --> 00:10:09,980 And in fact, in your problem set, 205 00:10:09,980 --> 00:10:14,630 you had all of these different cholesterol analogs. 206 00:10:14,630 --> 00:10:17,060 I mean, I think we still really don't get it. 207 00:10:17,060 --> 00:10:19,320 That's complicated-- we talked about this in class. 208 00:10:19,320 --> 00:10:21,830 You have these transmembrane helices-- 209 00:10:21,830 --> 00:10:25,310 what is it that's actually the signaling agent? 210 00:10:25,310 --> 00:10:28,670 So people are still asking that question, 211 00:10:28,670 --> 00:10:32,460 and we haven't quite gotten that far. 212 00:10:32,460 --> 00:10:38,480 But if you've read the reading, for HMG CoA reductase 213 00:10:38,480 --> 00:10:40,820 degradation, which is what we we're 214 00:10:40,820 --> 00:10:42,350 going to be talking about in class, 215 00:10:42,350 --> 00:10:47,600 the signaler is not the sterile, it's lanosterol. 216 00:10:47,600 --> 00:10:49,520 OK, and where have you seen lanosterol? 217 00:10:49,520 --> 00:10:52,940 The biosynthetic pathway has lanosterol 218 00:10:52,940 --> 00:10:54,270 sitting in the middle. 219 00:10:54,270 --> 00:10:58,130 It's not all that different, structurally, from cholesterol. 220 00:10:58,130 --> 00:11:01,760 You need to go back in, they all have four-membered rings, 221 00:11:01,760 --> 00:11:06,060 they have different extra methyl groups. 222 00:11:06,060 --> 00:11:08,900 So people are trying to sort that out. 223 00:11:08,900 --> 00:11:10,580 I don't think we really know. 224 00:11:10,580 --> 00:11:11,150 But how well? 225 00:11:11,150 --> 00:11:13,954 So you're right, they use sterols. 226 00:11:13,954 --> 00:11:16,370 They didn't use that, they didn't see very much difference 227 00:11:16,370 --> 00:11:17,120 with the sterols. 228 00:11:17,120 --> 00:11:20,450 What was the other way, which is sort of unusual, 229 00:11:20,450 --> 00:11:25,010 that they added cholesterol back into the system. 230 00:11:25,010 --> 00:11:29,660 So they could add it back with the methyl cyclodextrin-- 231 00:11:29,660 --> 00:11:32,380 they told you that that worked, and if you believe that-- 232 00:11:32,380 --> 00:11:35,438 and you look at the data-- it looked like that was happening. 233 00:11:39,920 --> 00:11:41,090 Nobody remembers? 234 00:11:41,090 --> 00:11:44,890 OK, well, we'll get to that in a little bit. 235 00:11:44,890 --> 00:11:47,720 OK, so the question we're focusing on 236 00:11:47,720 --> 00:11:50,570 is what are the changes in concentrations 237 00:11:50,570 --> 00:11:53,030 of cholesterol in the ER? 238 00:11:53,030 --> 00:11:58,520 So what method did they use to try 239 00:11:58,520 --> 00:12:03,320 to separate the ER membranes from all the other membranes? 240 00:12:05,948 --> 00:12:08,510 AUDIENCE: They first separated the [INAUDIBLE]---- 241 00:12:08,510 --> 00:12:10,804 JOANNE STUBBE: They separated the what? 242 00:12:10,804 --> 00:12:15,524 AUDIENCE: The sterols and the nucleus in the [INAUDIBLE].. 243 00:12:15,524 --> 00:12:16,940 JOANNE STUBBE: OK, so that's good. 244 00:12:16,940 --> 00:12:18,900 You can separate out the nucleus, 245 00:12:18,900 --> 00:12:21,920 and you could do that by ultracentrifugation-- we've 246 00:12:21,920 --> 00:12:25,520 seen that used in different kinds of ultracentrifugation. 247 00:12:25,520 --> 00:12:30,600 We've seen the different particles, 248 00:12:30,600 --> 00:12:36,290 the lipoproteins in the diet, how do we separate those? 249 00:12:36,290 --> 00:12:38,462 We talked about that in class briefly, 250 00:12:38,462 --> 00:12:39,920 you haven't had any papers to read. 251 00:12:39,920 --> 00:12:42,110 But what was the method of separation? 252 00:12:42,110 --> 00:12:43,800 If you look at all those particles-- 253 00:12:43,800 --> 00:12:46,820 remember we had a little cartoon of all the particles, 254 00:12:46,820 --> 00:12:50,400 and we focused on LDL, which is the particle that 255 00:12:50,400 --> 00:12:51,690 has the most cholesterol. 256 00:12:51,690 --> 00:12:53,960 So that's why everybody is focusing on that. 257 00:12:53,960 --> 00:12:55,970 What was the basis of the separation? 258 00:12:55,970 --> 00:12:57,730 AUDIENCE: Was it sucrose screening? 259 00:12:57,730 --> 00:12:59,420 JOANNE STUBBE: Was the what? 260 00:12:59,420 --> 00:13:00,260 AUDIENCE: Was it a sucrose screening-- 261 00:13:00,260 --> 00:13:01,100 the ultracentrifugation? 262 00:13:01,100 --> 00:13:02,266 JOANNE STUBBE: You need to-- 263 00:13:02,266 --> 00:13:04,200 AUDIENCE: Did they use a sucrose screening, 264 00:13:04,200 --> 00:13:05,532 like ultracentrifugation? 265 00:13:05,532 --> 00:13:07,240 JOANNE STUBBE: Yeah, ultracentrifugation. 266 00:13:07,240 --> 00:13:09,250 But how did the-- 267 00:13:09,250 --> 00:13:10,750 AUDIENCE: For the sucrose screening? 268 00:13:10,750 --> 00:13:13,041 JOANNE STUBBE: Yeah, OK, so they have different density 269 00:13:13,041 --> 00:13:15,520 gradients. , OK so that's going to be a key thing, 270 00:13:15,520 --> 00:13:18,084 and that's because if you look at the composition, 271 00:13:18,084 --> 00:13:19,750 they have different amounts of proteins, 272 00:13:19,750 --> 00:13:21,250 different amounts of fats. 273 00:13:21,250 --> 00:13:23,930 And they have different-- they float differently. 274 00:13:23,930 --> 00:13:27,520 So that's the method that they're going to use here. 275 00:13:27,520 --> 00:13:28,510 Is that a good method? 276 00:13:28,510 --> 00:13:30,810 Can you think of a better method? 277 00:13:30,810 --> 00:13:35,690 So in order to understand the switch for cholesterol, 278 00:13:35,690 --> 00:13:39,130 you've got to be able to measure the changes in cholesterol. 279 00:13:39,130 --> 00:13:42,160 Not an easy problem, because cholesterol is 280 00:13:42,160 --> 00:13:45,520 really insoluble in everything. 281 00:13:45,520 --> 00:13:48,190 And so how much is really in there, 282 00:13:48,190 --> 00:13:51,330 and how does it change under different sets of conditions? 283 00:13:51,330 --> 00:13:53,020 So is this a good method? 284 00:13:53,020 --> 00:13:53,830 What do you think? 285 00:13:53,830 --> 00:13:56,526 We'll look at the method in a little more detail, when 286 00:13:56,526 --> 00:13:58,150 I pull up the figures, but what did you 287 00:13:58,150 --> 00:14:01,540 think when you read the paper? 288 00:14:04,991 --> 00:14:07,456 AUDIENCE: Seems a pretty good method, 289 00:14:07,456 --> 00:14:12,386 other than that they're slightly different 290 00:14:12,386 --> 00:14:20,128 any other like properties different from the membrane 291 00:14:20,128 --> 00:14:21,753 than say, press on golgi bodies and ER. 292 00:14:21,753 --> 00:14:23,750 So it's like the only one I can think of. 293 00:14:23,750 --> 00:14:26,460 JOANNE STUBBE: Yeah, so the question is, you 294 00:14:26,460 --> 00:14:28,230 could you separate? 295 00:14:28,230 --> 00:14:32,660 Even separating the nucleus from the cytosol is not so trivial. 296 00:14:32,660 --> 00:14:35,010 But these methods are really gross methods, 297 00:14:35,010 --> 00:14:39,120 and during the centrifugation, things diffuse. 298 00:14:39,120 --> 00:14:40,890 So if you're having close separations, 299 00:14:40,890 --> 00:14:43,420 it's a equlibrating down this thing. 300 00:14:43,420 --> 00:14:46,070 And so you're getting your proteins, 301 00:14:46,070 --> 00:14:48,420 or your lipids are spreading out. 302 00:14:48,420 --> 00:14:52,680 Is there anything else any of you experience with insoluble-- 303 00:14:52,680 --> 00:14:55,770 this is what we're dealing with, is an insoluble mess, 304 00:14:55,770 --> 00:15:00,150 and how do you how do you separate things in a way 305 00:15:00,150 --> 00:15:02,190 that you have control over it so that you 306 00:15:02,190 --> 00:15:04,830 can address the key questions in this paper? 307 00:15:09,810 --> 00:15:12,170 Nobody thought about anything else? 308 00:15:12,170 --> 00:15:14,300 Did you like this method? 309 00:15:14,300 --> 00:15:17,862 Were you convinced by the data? 310 00:15:17,862 --> 00:15:20,352 AUDIENCE: I mean, like I couldn't necessarily 311 00:15:20,352 --> 00:15:22,842 think of something better. 312 00:15:22,842 --> 00:15:25,830 I don't know, I guess the thing that 313 00:15:25,830 --> 00:15:29,814 sketches me out the most about it just like how-- 314 00:15:29,814 --> 00:15:32,215 I'm not really familiar with the method. 315 00:15:32,215 --> 00:15:33,798 I haven't done this myself, so I don't 316 00:15:33,798 --> 00:15:37,734 know how that process affects the membrane integrity. 317 00:15:37,734 --> 00:15:40,150 JOANNE STUBBE: So that's an incredibly important question, 318 00:15:40,150 --> 00:15:42,880 because lipids confuse. 319 00:15:42,880 --> 00:15:43,879 They can mix. 320 00:15:43,879 --> 00:15:46,420 The question is, what are the rate constants for all of that? 321 00:15:46,420 --> 00:15:48,640 And we don't really teach very much 322 00:15:48,640 --> 00:15:50,740 in the introductory courses about lipids, 323 00:15:50,740 --> 00:15:55,060 and they're partitioning between other membranes and fusion, 324 00:15:55,060 --> 00:15:55,910 and all that stuff. 325 00:15:55,910 --> 00:15:58,780 But if you think about it, that's what the cell is, right? 326 00:15:58,780 --> 00:16:00,670 How do you get a plasma membrane, 327 00:16:00,670 --> 00:16:03,530 and all these membranes around all these little organelles-- 328 00:16:03,530 --> 00:16:06,100 that's an amazing observation. 329 00:16:06,100 --> 00:16:08,200 And we've seen in class already, what 330 00:16:08,200 --> 00:16:13,990 have we seen to get LDL receptor from here to the plasma 331 00:16:13,990 --> 00:16:15,090 membrane? 332 00:16:15,090 --> 00:16:17,710 How do we have to do that? 333 00:16:17,710 --> 00:16:20,770 We had to use these little vesicles. 334 00:16:20,770 --> 00:16:24,020 So you're generating something over here, 335 00:16:24,020 --> 00:16:26,310 it goes through the Golgi stack. 336 00:16:26,310 --> 00:16:28,540 Again, another set of membranes has 337 00:16:28,540 --> 00:16:31,810 got to come out the different levels of the Golgi stack. 338 00:16:31,810 --> 00:16:37,360 And then it's still got to get into the plasma membrane, 339 00:16:37,360 --> 00:16:39,730 and fuse, and dump its cargo. 340 00:16:39,730 --> 00:16:42,610 So I think it's an amazing process. 341 00:16:42,610 --> 00:16:44,110 And people interested in evolution, 342 00:16:44,110 --> 00:16:46,630 this is one of the major things people are focused on 343 00:16:46,630 --> 00:16:51,760 is, how can you make cells, little fake cells, 344 00:16:51,760 --> 00:16:53,950 artificial cells, that can replicate themselves. 345 00:16:53,950 --> 00:16:55,366 You can make it, and they're going 346 00:16:55,366 --> 00:16:57,820 to have to divide and fuse. 347 00:16:57,820 --> 00:17:00,520 And it's exactly the same problem here. 348 00:17:00,520 --> 00:17:04,569 And so this question of fluidity is an extremely important 349 00:17:04,569 --> 00:17:05,079 question. 350 00:17:05,079 --> 00:17:07,960 And a lot of people that focus on lipids-- 351 00:17:07,960 --> 00:17:12,619 which is not a popular thing to study, because it's so hard-- 352 00:17:12,619 --> 00:17:14,170 it's incredibly important. 353 00:17:14,170 --> 00:17:17,410 And people that look at membrane proteins, 354 00:17:17,410 --> 00:17:20,440 they almost always have lipids on them. 355 00:17:20,440 --> 00:17:22,060 And when you do them yourself, you 356 00:17:22,060 --> 00:17:25,300 have a detergent, which is not a real lipid-- 357 00:17:25,300 --> 00:17:26,660 does that change the property? 358 00:17:26,660 --> 00:17:28,630 So all of these questions, I think, 359 00:17:28,630 --> 00:17:32,110 are really central to what happens 360 00:17:32,110 --> 00:17:36,860 in the membranes, which is a lot of stuff inside the cell. 361 00:17:36,860 --> 00:17:40,000 So I think it's good to question what they did. 362 00:17:40,000 --> 00:17:43,730 I think their results turned out to be quite interesting. 363 00:17:43,730 --> 00:17:45,040 But we'll come back-- 364 00:17:45,040 --> 00:17:46,840 I think that was a hard problem. 365 00:17:46,840 --> 00:17:49,340 And so we'll come back and we'll look at this. 366 00:17:49,340 --> 00:17:56,050 And so then, let's say that we could end up separating things. 367 00:17:56,050 --> 00:18:01,390 Then the question is, what was the key type of measurement 368 00:18:01,390 --> 00:18:07,600 they made, where they could correlate the changes 369 00:18:07,600 --> 00:18:08,800 in cholesterol levels-- 370 00:18:08,800 --> 00:18:11,470 we talked about, you can control perhaps the cholesterol 371 00:18:11,470 --> 00:18:14,970 levels with the cyclodextrin. 372 00:18:14,970 --> 00:18:18,310 But then, how did they correlate the changes 373 00:18:18,310 --> 00:18:20,820 in the cholesterol levels in the membrane 374 00:18:20,820 --> 00:18:25,570 with this transcriptional regulation? 375 00:18:25,570 --> 00:18:30,310 Which, that is what happens with the steroid-responsive 376 00:18:30,310 --> 00:18:32,870 element-binding protein, the transcription factor. 377 00:18:32,870 --> 00:18:36,350 So what happens in that process? 378 00:18:36,350 --> 00:18:41,770 What are the changes in the SRE BP 379 00:18:41,770 --> 00:18:45,610 dependent on the concentrations of the cholesterol? 380 00:18:45,610 --> 00:18:48,150 And how did they take advantage of that 381 00:18:48,150 --> 00:18:53,830 in answering this question about what the cholesterol 382 00:18:53,830 --> 00:18:58,870 levels were that allowed you to turn on transcription 383 00:18:58,870 --> 00:19:03,440 of LDL receptor, and HMG CoA reductase. 384 00:19:03,440 --> 00:19:04,750 So what's the major assay? 385 00:19:04,750 --> 00:19:06,937 We'll look at that, as well. 386 00:19:06,937 --> 00:19:08,770 So if you go back and you look at the model, 387 00:19:08,770 --> 00:19:09,985 what happens in this model? 388 00:19:15,170 --> 00:19:17,050 All right, here we go-- 389 00:19:17,050 --> 00:19:18,610 what happens in this model? 390 00:19:18,610 --> 00:19:20,665 What's happening to SREBP? 391 00:19:23,395 --> 00:19:25,375 AUDIENCE: It has completely changed 392 00:19:25,375 --> 00:19:28,345 and exposed [INAUDIBLE]. 393 00:19:28,345 --> 00:19:30,700 JOANNE STUBBE: No, that's SCAP-- 394 00:19:30,700 --> 00:19:33,140 SCAP, that's this guy. 395 00:19:33,140 --> 00:19:33,970 OK? 396 00:19:33,970 --> 00:19:35,714 So SCAP, that's a key player. 397 00:19:35,714 --> 00:19:36,880 That's what we talked about. 398 00:19:36,880 --> 00:19:38,890 I know the names are all confusing. 399 00:19:38,890 --> 00:19:41,260 You're going to need to write these down to remember. 400 00:19:41,260 --> 00:19:42,920 The names are very confusing. 401 00:19:42,920 --> 00:19:43,653 Yeah? 402 00:19:43,653 --> 00:19:48,090 AUDIENCE: So the SCAP SREBP, whatever you call it, 403 00:19:48,090 --> 00:19:54,840 complex move signal g-apperatus then part of it's cleaved 404 00:19:54,840 --> 00:19:56,120 and moves to the nucleus? 405 00:19:56,120 --> 00:19:58,150 JOANNE STUBBE: Right, so how could you 406 00:19:58,150 --> 00:19:59,710 take advantage of that? 407 00:19:59,710 --> 00:20:03,700 This is the key observation that they're taking advantage of, 408 00:20:03,700 --> 00:20:05,140 to ask the question-- 409 00:20:05,140 --> 00:20:08,740 since this whole process is dependent on the concentration 410 00:20:08,740 --> 00:20:09,750 of cholesterol. 411 00:20:09,750 --> 00:20:11,320 If you have high cholesterol, there's 412 00:20:11,320 --> 00:20:14,650 no way you want this to happen-- you want to shut it off. 413 00:20:14,650 --> 00:20:19,060 If you have low cholesterol, you want to turn these guys on. 414 00:20:19,060 --> 00:20:22,480 So this movement is the key. 415 00:20:22,480 --> 00:20:24,460 And what do we see, if we look at what 416 00:20:24,460 --> 00:20:27,467 happens to this protein, SREBP, what happens to it 417 00:20:27,467 --> 00:20:28,300 during this process? 418 00:20:30,970 --> 00:20:31,830 It gets cleaved. 419 00:20:31,830 --> 00:20:34,990 And how could you monitor that cleavage? 420 00:20:34,990 --> 00:20:37,218 How do they do it in the paper? 421 00:20:37,218 --> 00:20:40,122 AUDIENCE: They used a-- 422 00:20:40,122 --> 00:20:42,574 was it a [? florifor-- ?] or is that the homework? 423 00:20:42,574 --> 00:20:44,740 JOANNE STUBBE: They could use a [? florifor, ?] they 424 00:20:44,740 --> 00:20:46,580 didn't do that. 425 00:20:46,580 --> 00:20:47,610 They did a what? 426 00:20:50,508 --> 00:20:58,154 AUDIENCE: They were able to separate the [INAUDIBLE] gel? 427 00:20:58,154 --> 00:21:00,070 JOANNE STUBBE: So it can be operated by a gel. 428 00:21:00,070 --> 00:21:03,840 So to me, this is quite an easy assay. 429 00:21:03,840 --> 00:21:05,100 Because if you look at this-- 430 00:21:05,100 --> 00:21:07,050 I don't remember what the molecular weight is, 431 00:21:07,050 --> 00:21:10,180 but it's a lot smaller over here. 432 00:21:10,180 --> 00:21:14,100 And so, that turns out to be a great assay. 433 00:21:14,100 --> 00:21:18,630 So that part of their analysis, I think, 434 00:21:18,630 --> 00:21:21,100 was a really smart part of the analysis. 435 00:21:21,100 --> 00:21:23,620 And so then the question becomes, 436 00:21:23,620 --> 00:21:26,320 can you quantitate all of this? 437 00:21:26,320 --> 00:21:28,680 So if you have a lot of cholesterol, 438 00:21:28,680 --> 00:21:29,790 this doesn't happen. 439 00:21:29,790 --> 00:21:35,100 And so everything is bigger, and resides in the membrane. 440 00:21:35,100 --> 00:21:37,420 You could even probably look at that. 441 00:21:37,420 --> 00:21:43,350 Whereas, when the cholesterol is really lower, things go there. 442 00:21:43,350 --> 00:21:44,730 And it's everything in between. 443 00:21:44,730 --> 00:21:46,660 The question is, what is the concept-- 444 00:21:46,660 --> 00:21:50,820 can you measure if you have X% cholesterol in the ER, 445 00:21:50,820 --> 00:21:55,620 how much do you have to decrease it to see a change or a switch 446 00:21:55,620 --> 00:21:58,260 in where this protein goes? 447 00:21:58,260 --> 00:22:02,070 So I think the experimental design is actually 448 00:22:02,070 --> 00:22:04,320 amazingly creative. 449 00:22:04,320 --> 00:22:07,990 But then you see the data of the other side. 450 00:22:07,990 --> 00:22:13,680 And what I want to do now is focus on what the issues are. 451 00:22:13,680 --> 00:22:17,640 So we're going to come back and look at, 452 00:22:17,640 --> 00:22:19,920 how did they look at SREBP? 453 00:22:19,920 --> 00:22:23,040 So you could look at this a number 454 00:22:23,040 --> 00:22:28,280 of ways-- you could look at this by protein gel directly. 455 00:22:28,280 --> 00:22:32,040 How else do people look at proteins using westerns? 456 00:22:32,040 --> 00:22:34,560 What's a western? 457 00:22:34,560 --> 00:22:37,165 Anybody know what a western analysis is? 458 00:22:37,165 --> 00:22:39,165 Didn't I ask you that at the beginning of class? 459 00:22:42,430 --> 00:22:44,170 How else do you detect proteins? 460 00:22:44,170 --> 00:22:48,020 You've seen this in the first half of the semester a lot. 461 00:22:48,020 --> 00:22:49,130 Yeah, antibodies. 462 00:22:49,130 --> 00:22:51,630 So if you have antibodies to this-- 463 00:22:51,630 --> 00:22:54,670 and we'll talk about this, because the western analysis, 464 00:22:54,670 --> 00:22:58,180 which people use all the time, and there are so 465 00:22:58,180 --> 00:22:59,860 many issues with it, that I think 466 00:22:59,860 --> 00:23:03,850 I want you to think about what the issues are. 467 00:23:03,850 --> 00:23:06,400 And then you correlate the two-- 468 00:23:06,400 --> 00:23:07,960 changing the levels of cholesterol. 469 00:23:07,960 --> 00:23:12,340 Which they measure by mass spec after separation 470 00:23:12,340 --> 00:23:15,550 and purification of lipids, and the cleavage. 471 00:23:15,550 --> 00:23:17,920 And they plot the data, and that's 472 00:23:17,920 --> 00:23:20,500 where they got the analysis from. 473 00:23:20,500 --> 00:23:23,140 So the first thing that you want to do-- the first thing, 474 00:23:23,140 --> 00:23:27,370 and the key to everything, is separation of the membranes. 475 00:23:27,370 --> 00:23:31,960 And so, this is a cartoon of when you put something, 476 00:23:31,960 --> 00:23:35,070 you load something on the top, and you have a gradient, 477 00:23:35,070 --> 00:23:37,630 and the gradient could be made of a number of things. 478 00:23:37,630 --> 00:23:40,500 Have any of you ever run these kinds of gradients? 479 00:23:40,500 --> 00:23:43,000 OK, so you can make them out of glycerol, 480 00:23:43,000 --> 00:23:44,530 you can make them out of sucrose-- 481 00:23:44,530 --> 00:23:46,840 did anybody look at how these gradients were made? 482 00:23:46,840 --> 00:23:48,814 Did you read the experimental carefully enough 483 00:23:48,814 --> 00:23:49,480 to look at that? 484 00:23:54,720 --> 00:23:56,605 Yeah, how do you make a sucrose gradient? 485 00:24:02,790 --> 00:24:03,670 You have no idea? 486 00:24:03,670 --> 00:24:06,050 But yeah, so layering. 487 00:24:06,050 --> 00:24:08,709 So what you really like to do is have a continuous gradient, 488 00:24:08,709 --> 00:24:09,250 or something. 489 00:24:09,250 --> 00:24:11,920 But sucrose is incredibly viscous. 490 00:24:11,920 --> 00:24:14,020 So if you were trying to make a linear gradient, 491 00:24:14,020 --> 00:24:16,122 which you could do by mixing two things 492 00:24:16,122 --> 00:24:18,580 of different concentrations-- if you could get them to stir 493 00:24:18,580 --> 00:24:20,947 really well, and then add it in, and you 494 00:24:20,947 --> 00:24:22,030 could generate a gradient. 495 00:24:22,030 --> 00:24:24,850 But it's so hard to do, that what happens 496 00:24:24,850 --> 00:24:26,050 is they end up layering it. 497 00:24:26,050 --> 00:24:31,150 So they make X%, Y%, Z%, they put it down. 498 00:24:31,150 --> 00:24:33,610 And then they try to layer something on top of it. 499 00:24:33,610 --> 00:24:38,170 And then they put whatever the interest in at the top, 500 00:24:38,170 --> 00:24:40,930 and then they centrifuge it. 501 00:24:40,930 --> 00:24:43,960 So what are the issues? 502 00:24:43,960 --> 00:24:48,484 Do you think this is what the gradient would look like? 503 00:24:48,484 --> 00:24:49,900 So what are the issues when you're 504 00:24:49,900 --> 00:24:54,090 doing this, when you layer it? 505 00:24:54,090 --> 00:24:56,050 And this is why the data-- 506 00:24:56,050 --> 00:24:58,410 which we'll talk about in a minute-- 507 00:24:58,410 --> 00:25:01,684 is the data, or part of the issue is this method. 508 00:25:01,684 --> 00:25:03,600 That's why you need to think about the method. 509 00:25:03,600 --> 00:25:05,370 And there are better ways to do this. 510 00:25:05,370 --> 00:25:09,000 And it really depends on what you're trying to separate. 511 00:25:09,000 --> 00:25:11,850 So if this band-- 512 00:25:11,850 --> 00:25:14,130 say these were two bands, you wouldn't really 513 00:25:14,130 --> 00:25:15,676 get very much separation at all. 514 00:25:15,676 --> 00:25:17,050 If there were two separate things 515 00:25:17,050 --> 00:25:21,870 that sedimented under these conditions very close together. 516 00:25:21,870 --> 00:25:24,460 So what would happen when you're sedimenting this? 517 00:25:24,460 --> 00:25:26,750 Does anybody have any idea how long it takes? 518 00:25:26,750 --> 00:25:29,040 Do you think you'd do this in a centrifuge, 519 00:25:29,040 --> 00:25:30,810 you spin it for three minutes, and then-- 520 00:25:33,460 --> 00:25:37,620 so sometimes you sediment these things for 16, 20 hours. 521 00:25:37,620 --> 00:25:41,790 So what happens during the sedimentation? 522 00:25:41,790 --> 00:25:44,520 That might make this more challenging, 523 00:25:44,520 --> 00:25:46,892 in terms of separating what you want to separate? 524 00:25:46,892 --> 00:25:49,100 AUDIENCE: I'm not sure, but it [INAUDIBLE] diffusion. 525 00:25:49,100 --> 00:25:51,450 JOANNE STUBBE: Yeah, so exactly, you have diffusion. 526 00:25:51,450 --> 00:25:54,330 And even when you've layered things on top of each other 527 00:25:54,330 --> 00:25:57,030 like that, you start to have diffusion. 528 00:25:57,030 --> 00:26:01,030 And if you shake up the tube a little bit, it's all over. 529 00:26:01,030 --> 00:26:03,720 So how do you prepare these things is not-- 530 00:26:03,720 --> 00:26:05,790 so people still use these methods, 531 00:26:05,790 --> 00:26:08,220 but I would like to see better methods. 532 00:26:08,220 --> 00:26:12,270 And so they tried one method with sucrose, 533 00:26:12,270 --> 00:26:14,130 and then that wasn't good enough. 534 00:26:14,130 --> 00:26:15,150 We'll look at the data. 535 00:26:15,150 --> 00:26:17,630 So they went to a second method. 536 00:26:17,630 --> 00:26:20,007 And where did they come up with this? 537 00:26:20,007 --> 00:26:21,840 I have no idea where they came up with this, 538 00:26:21,840 --> 00:26:24,840 but there was an MD PhD student in our class 539 00:26:24,840 --> 00:26:27,330 who had seen this and one of his classes, 540 00:26:27,330 --> 00:26:29,280 and they use it and some blood test. 541 00:26:29,280 --> 00:26:31,950 So I think that's probably where these guys got it from, 542 00:26:31,950 --> 00:26:35,310 because Brown and Goldstein are both MDs. 543 00:26:35,310 --> 00:26:38,220 But again, it's just another way to make a gradient. 544 00:26:38,220 --> 00:26:42,090 And I'm not sure why this gradient works 545 00:26:42,090 --> 00:26:45,960 as effectively as it does. 546 00:26:45,960 --> 00:26:48,520 But the first gradient didn't work so great, 547 00:26:48,520 --> 00:26:49,770 and we'll look at that data. 548 00:26:49,770 --> 00:26:52,290 So then they added on a few more steps, 549 00:26:52,290 --> 00:26:57,040 because they weren't happy with the level of separation. 550 00:26:57,040 --> 00:26:59,640 So looking at membranes, I think this 551 00:26:59,640 --> 00:27:02,070 is going to be more and more looking at membranes, 552 00:27:02,070 --> 00:27:04,910 because membranes, you have two leaflets-- 553 00:27:04,910 --> 00:27:07,020 the lipids and the leaflets are different. 554 00:27:07,020 --> 00:27:10,170 Do you think that affects the biology? 555 00:27:10,170 --> 00:27:12,870 I guarantee you it affects the biology in ways 556 00:27:12,870 --> 00:27:15,510 that we would really like to understand that I don't 557 00:27:15,510 --> 00:27:18,180 think we understand very well. 558 00:27:18,180 --> 00:27:20,910 When you isolate a membrane protein, have any of you 559 00:27:20,910 --> 00:27:24,280 ever isolated a membrane protein? 560 00:27:24,280 --> 00:27:25,950 So you have an insoluble-- 561 00:27:25,950 --> 00:27:28,945 it's in this lipid system. 562 00:27:28,945 --> 00:27:30,570 How do you think you get it out, so you 563 00:27:30,570 --> 00:27:34,680 can go through the steps, a protein purification 564 00:27:34,680 --> 00:27:37,680 that you've talked about, or you have probably done 565 00:27:37,680 --> 00:27:40,550 in an introductory lab course? 566 00:27:40,550 --> 00:27:42,360 What is the first thing you need to do? 567 00:27:49,122 --> 00:27:50,370 Yeah, solubalize it. 568 00:27:50,370 --> 00:27:51,995 And how do you solubalize it? 569 00:27:51,995 --> 00:27:53,120 AUDIENCE: With a detergent. 570 00:27:53,120 --> 00:27:55,161 JOANNE STUBBE: Yeah, with some kind of detergent. 571 00:27:55,161 --> 00:27:58,310 It's like what you saw with a kilo microns, or the bile acids 572 00:27:58,310 --> 00:27:59,640 that we talked about. 573 00:27:59,640 --> 00:28:01,700 So you can use different-- and people have 574 00:28:01,700 --> 00:28:03,500 their own favorite detergents. 575 00:28:03,500 --> 00:28:06,990 But again, that changes things. 576 00:28:06,990 --> 00:28:09,890 But otherwise, you can't purify anything 577 00:28:09,890 --> 00:28:13,120 unless you happen to have a membrane where 578 00:28:13,120 --> 00:28:14,739 the only protein in the membrane is 579 00:28:14,739 --> 00:28:17,030 the one you're interested in, which, of course, doesn't 580 00:28:17,030 --> 00:28:18,530 exist. 581 00:28:18,530 --> 00:28:20,090 So anyhow, they went through that. 582 00:28:20,090 --> 00:28:22,410 And then what did they end up seeing? 583 00:28:22,410 --> 00:28:25,430 So they went through different steps, 584 00:28:25,430 --> 00:28:32,941 and they separate them into different-- the supernate, 585 00:28:32,941 --> 00:28:36,590 or the light and the heavy membrane fractions. 586 00:28:36,590 --> 00:28:38,072 And then they have to analyze it. 587 00:28:38,072 --> 00:28:39,530 And so the question is, how do they 588 00:28:39,530 --> 00:28:45,230 analyze to tell how well these separations actually worked? 589 00:28:45,230 --> 00:28:49,930 What was the method that they did 590 00:28:49,930 --> 00:28:53,000 to determine whether they separated 591 00:28:53,000 --> 00:28:57,200 the ER from the plasma membrane, from the Golgi stacks, 592 00:28:57,200 --> 00:29:00,410 from the lisosomes, from the peroxisomes. 593 00:29:00,410 --> 00:29:03,320 So they have all we have all these little organelles 594 00:29:03,320 --> 00:29:04,130 in there. 595 00:29:04,130 --> 00:29:09,800 What did they do to test each one of these fractions? 596 00:29:09,800 --> 00:29:15,560 Let me ask you this question-- how do you think they got the-- 597 00:29:15,560 --> 00:29:20,360 how do you how did they get the material out of these gradients 598 00:29:20,360 --> 00:29:24,180 to do the experiments that I was just talking about. 599 00:29:24,180 --> 00:29:27,350 So they want to analyze what's in each of these bands. 600 00:29:27,350 --> 00:29:30,668 How did they get it out of this tube? 601 00:29:30,668 --> 00:29:32,419 AUDIENCE: Would they use a Pasteur filter? 602 00:29:32,419 --> 00:29:33,918 JOANNE STUBBE: So what do you think? 603 00:29:33,918 --> 00:29:35,890 You just stick it down in and suck it out? 604 00:29:35,890 --> 00:29:38,020 Well, I mean, yes, so what do you think? 605 00:29:38,020 --> 00:29:39,400 You could do that-- 606 00:29:39,400 --> 00:29:42,820 you open the top, you stick it in, you carefully stick it in. 607 00:29:42,820 --> 00:29:44,050 If you can see it. 608 00:29:44,050 --> 00:29:47,200 Lots of times you can see these lipids, because they're opaque, 609 00:29:47,200 --> 00:29:47,780 or something. 610 00:29:47,780 --> 00:29:48,580 So you can see. 611 00:29:48,580 --> 00:29:53,440 Or, if you still hope your sucrose layers, lots of times 612 00:29:53,440 --> 00:29:56,050 they layer in between the different concentrations 613 00:29:56,050 --> 00:29:59,170 of the sucrose, and you see white stuff precipitating. 614 00:29:59,170 --> 00:30:02,590 So you could conceivably stick a pipe head from the top 615 00:30:02,590 --> 00:30:03,940 and suck it out. 616 00:30:03,940 --> 00:30:05,410 AUDIENCE: But that would perturb all the other layers. 617 00:30:05,410 --> 00:30:06,250 JOANNE STUBBE: Absolutely it would 618 00:30:06,250 --> 00:30:07,526 perturb all the other layers. 619 00:30:07,526 --> 00:30:09,400 So here you're doing something-- it's already 620 00:30:09,400 --> 00:30:11,858 a very hard experiment, because they're all being perturbed 621 00:30:11,858 --> 00:30:14,190 anyhow, because of diffusion. 622 00:30:14,190 --> 00:30:16,450 So is there any other way you could think 623 00:30:16,450 --> 00:30:19,850 about separating these things? 624 00:30:19,850 --> 00:30:23,530 And so, the hint is that they use plastic tubes. 625 00:30:27,880 --> 00:30:29,760 So these things are not glass. 626 00:30:29,760 --> 00:30:30,820 Most centrifuges-- 627 00:30:30,820 --> 00:30:31,690 AUDIENCE: Freeze it? 628 00:30:31,690 --> 00:30:32,560 Cut it? 629 00:30:32,560 --> 00:30:35,540 JOANNE STUBBE: Well, so you don't do that, that could be-- 630 00:30:35,540 --> 00:30:37,320 OK, so you could. 631 00:30:37,320 --> 00:30:39,640 But you then have to, if you were cutting it, 632 00:30:39,640 --> 00:30:42,460 you still have to get it out of the tube. 633 00:30:42,460 --> 00:30:46,360 Unless you had a saw that didn't have any vibrations when 634 00:30:46,360 --> 00:30:49,270 you were cutting it, of course, which would not happen. 635 00:30:49,270 --> 00:30:52,690 But if you look here in this cartoon, 636 00:30:52,690 --> 00:30:54,700 so I gave you this, what are they doing here? 637 00:30:54,700 --> 00:30:58,390 They're sticking a syringe in through the side of the tube. 638 00:30:58,390 --> 00:31:00,530 And that's still what people use. 639 00:31:00,530 --> 00:31:03,240 So you can suck out-- if you can see something. 640 00:31:03,240 --> 00:31:05,170 So you have to be able to see in some way 641 00:31:05,170 --> 00:31:09,685 to know where to suck it out, so you might have a way, actually, 642 00:31:09,685 --> 00:31:12,250 in doing ultracentrifugations. 643 00:31:12,250 --> 00:31:14,870 I think with the lipids you can see them by eyeball, 644 00:31:14,870 --> 00:31:18,670 but you might look at absorption. 645 00:31:18,670 --> 00:31:23,560 If they have proteins, you could monitor absorption 646 00:31:23,560 --> 00:31:27,040 through the gradient, and that might tell you 647 00:31:27,040 --> 00:31:28,240 how to fractionate things. 648 00:31:28,240 --> 00:31:29,980 But anyhow, that's also an issue. 649 00:31:29,980 --> 00:31:33,790 Because before they can do the next step in the analysis, 650 00:31:33,790 --> 00:31:37,000 they've got to get the material out. 651 00:31:37,000 --> 00:31:40,660 So they've got the material out in each of these steps, 652 00:31:40,660 --> 00:31:42,930 and then, how do they look at this? 653 00:31:42,930 --> 00:31:45,970 They can pull it out. 654 00:31:45,970 --> 00:31:49,200 So what are they looking for? 655 00:31:49,200 --> 00:31:55,850 To tell them how effective this method is. 656 00:31:55,850 --> 00:31:59,230 AUDIENCE: Maybe some specific markers for each protein. 657 00:31:59,230 --> 00:32:00,280 JOANNE STUBBE: Exactly. 658 00:32:00,280 --> 00:32:03,640 So what are they-- 659 00:32:03,640 --> 00:32:05,860 to do that, what they're going to have to do 660 00:32:05,860 --> 00:32:10,450 is, before we look at the details of the method, 661 00:32:10,450 --> 00:32:12,760 I want to go through a western blot. 662 00:32:12,760 --> 00:32:15,087 So what do we know about a western blot? 663 00:32:15,087 --> 00:32:18,217 AUDIENCE: I have a quick question about the method here. 664 00:32:18,217 --> 00:32:19,800 JOANNE STUBBE: About the which method? 665 00:32:19,800 --> 00:32:21,383 AUDIENCE: The lysis method [INAUDIBLE] 666 00:32:21,383 --> 00:32:23,162 ball bearing homogenizer. 667 00:32:23,162 --> 00:32:26,230 So they're literally putting these cells in something 668 00:32:26,230 --> 00:32:27,481 like a bunch of ball bearings? 669 00:32:27,481 --> 00:32:29,105 JOANNE STUBBE: Yeah, you could do that. 670 00:32:29,105 --> 00:32:30,860 There's a lot of ways to crack open cells. 671 00:32:30,860 --> 00:32:32,410 I don't know which one's the best-- 672 00:32:32,410 --> 00:32:34,799 mammalian cells are really easy to open. 673 00:32:34,799 --> 00:32:36,340 Sometimes what I like to do is freeze 674 00:32:36,340 --> 00:32:37,756 and thaw them-- sometimes you have 675 00:32:37,756 --> 00:32:41,100 like a little mortar and pestle, or something like that. 676 00:32:41,100 --> 00:32:45,310 But that's-- I mean, yeast cells, you roll them. 677 00:32:45,310 --> 00:32:47,804 You have to have enough cells so you can do something. 678 00:32:47,804 --> 00:32:49,470 If you only have a tiny amount of cells, 679 00:32:49,470 --> 00:32:51,219 it makes it really challenging with beads, 680 00:32:51,219 --> 00:32:53,664 because it covers the beads. 681 00:32:53,664 --> 00:32:56,330 AUDIENCE: Do you have any issues with any of the different types 682 00:32:56,330 --> 00:32:57,177 of membranes that-- 683 00:32:57,177 --> 00:32:58,510 JOANNE STUBBE: Sticking to that? 684 00:32:58,510 --> 00:32:59,110 Absolutely. 685 00:32:59,110 --> 00:33:01,520 I'm sure you have to look at all of that kind of stuff. 686 00:33:01,520 --> 00:33:04,180 So how you choose, that's an important thing 687 00:33:04,180 --> 00:33:06,460 to look at, how you choose to crack open the cells. 688 00:33:06,460 --> 00:33:09,490 And it's the same with bacterial cells-- 689 00:33:09,490 --> 00:33:11,780 there are three or four ways to crack open the cells. 690 00:33:11,780 --> 00:33:15,490 And I can tell you only one of them really works efficiently. 691 00:33:15,490 --> 00:33:18,550 And a lot of people, when they use some of the others, 692 00:33:18,550 --> 00:33:20,500 they do something and they assume it works, 693 00:33:20,500 --> 00:33:22,720 but they never check to see whether the cell 694 00:33:22,720 --> 00:33:24,520 walls have been cracked open. 695 00:33:24,520 --> 00:33:26,950 A lot of times they haven't, and so what you get out 696 00:33:26,950 --> 00:33:30,040 is very, very low levels of protein, 697 00:33:30,040 --> 00:33:32,230 because you haven't cracked open the cell. 698 00:33:32,230 --> 00:33:34,960 So figuring out-- mammalian cells are apparently, 699 00:33:34,960 --> 00:33:36,430 I haven't worked with those myself, 700 00:33:36,430 --> 00:33:39,040 but they're apparently much easier 701 00:33:39,040 --> 00:33:41,200 to disrupt than bacteria. 702 00:33:41,200 --> 00:33:43,970 Or if you look at fungi-- 703 00:33:43,970 --> 00:33:46,630 fungi are really hard to crack open, yeast. 704 00:33:49,280 --> 00:33:52,180 So anyhow, that's an important thing to look at. 705 00:33:52,180 --> 00:33:57,340 So every one of these things, again, the devil 706 00:33:57,340 --> 00:33:58,284 is in the details. 707 00:33:58,284 --> 00:33:59,950 But when you're doing your own research, 708 00:33:59,950 --> 00:34:02,650 it doesn't matter what method you're looking at. 709 00:34:02,650 --> 00:34:05,860 The first time around, you need to look at it in detail, 710 00:34:05,860 --> 00:34:08,409 and convince yourself that this is a good way 711 00:34:08,409 --> 00:34:10,449 to chase this down. 712 00:34:10,449 --> 00:34:12,592 And you look at it in detail the first time around. 713 00:34:12,592 --> 00:34:14,050 And when you convince yourself it's 714 00:34:14,050 --> 00:34:16,175 working really well, and doing what you want to do, 715 00:34:16,175 --> 00:34:17,698 then you just use it. 716 00:34:17,698 --> 00:34:18,739 And that's the end of it. 717 00:34:18,739 --> 00:34:21,197 You don't have to go back and keep thinking about this over 718 00:34:21,197 --> 00:34:22,520 and over again. 719 00:34:22,520 --> 00:34:26,560 So the method we're going to use is a western blot. 720 00:34:26,560 --> 00:34:31,090 So we've got this stuff out, and have you all 721 00:34:31,090 --> 00:34:32,790 run SDS page shells? 722 00:34:32,790 --> 00:34:39,587 OK, so SDS page shells separate proteins how? 723 00:34:39,587 --> 00:34:40,670 AUDIENCE: Based on size... 724 00:34:40,670 --> 00:34:41,794 JOANNE STUBBE: By the what? 725 00:34:41,794 --> 00:34:46,760 AUDIENCE: It separates into a a charge gradient, and then-- 726 00:34:46,760 --> 00:34:48,967 not a charge gradient, but-- 727 00:34:48,967 --> 00:34:50,719 JOANNE STUBBE: Not charge. 728 00:34:50,719 --> 00:34:53,000 AUDIENCE: That's what drives the protein, but... 729 00:34:53,000 --> 00:34:55,449 JOANNE STUBBE: Right, but it's based on size, 730 00:34:55,449 --> 00:34:57,030 because it's coded-- 731 00:34:57,030 --> 00:35:01,580 every protein ratio is coded with this detergent, sodium 732 00:35:01,580 --> 00:35:04,750 dodecyl sulfate, which makes them migrate pretty 733 00:35:04,750 --> 00:35:06,860 much like the molecular weight. 734 00:35:06,860 --> 00:35:09,220 But if you've done these, it's not exactly 735 00:35:09,220 --> 00:35:10,360 like the molecular weight. 736 00:35:10,360 --> 00:35:13,030 You can do standards where you know the molecular weight, 737 00:35:13,030 --> 00:35:14,950 you can do a standard curve, and then 738 00:35:14,950 --> 00:35:17,590 you see where your protein migrates. 739 00:35:17,590 --> 00:35:20,140 And sometimes they migrate a little faster, sometimes 740 00:35:20,140 --> 00:35:22,900 a little slower, but it's OK. 741 00:35:22,900 --> 00:35:26,440 So you run this, and then what do you do? 742 00:35:26,440 --> 00:35:29,020 Does anybody know what you do next, to do a western? 743 00:35:31,530 --> 00:35:33,626 AUDIENCE: You need to use the membrane to... 744 00:35:33,626 --> 00:35:35,250 JOANNE STUBBE: Right, so the next thing 745 00:35:35,250 --> 00:35:37,820 they did was they used-- 746 00:35:37,820 --> 00:35:41,070 I'm going to put all of these up-- so they transferred it 747 00:35:41,070 --> 00:35:43,050 to a membrane. 748 00:35:43,050 --> 00:35:47,520 And why did they have to transfer it to a membrane 749 00:35:47,520 --> 00:35:50,460 to do this analysis? 750 00:35:50,460 --> 00:35:51,770 This is an extra step. 751 00:35:51,770 --> 00:35:52,570 And it turns out-- 752 00:35:56,480 --> 00:35:58,640 we're going to look at an antibody interacting 753 00:35:58,640 --> 00:35:59,300 with a protein. 754 00:35:59,300 --> 00:36:01,520 Why don't we just look at the antibody interacting 755 00:36:01,520 --> 00:36:03,393 with the protein to start with? 756 00:36:03,393 --> 00:36:04,490 AUDIENCE: It doesn't have access to the protein. 757 00:36:04,490 --> 00:36:05,890 JOANNE STUBBE: Right, it doesn't have very good access. 758 00:36:05,890 --> 00:36:07,830 It's really not very efficient. 759 00:36:07,830 --> 00:36:12,230 So people found, pretty much by trial and error, 760 00:36:12,230 --> 00:36:14,660 that you needed to transfer this to a membrane. 761 00:36:14,660 --> 00:36:16,910 I mean, we have hundreds of kinds of membranes. 762 00:36:16,910 --> 00:36:20,240 How did they choose nitrocellulose? 763 00:36:20,240 --> 00:36:22,510 If any of you have one run westerns, 764 00:36:22,510 --> 00:36:25,340 you remember what kind of a membrane you used? 765 00:36:25,340 --> 00:36:28,490 Did you use nitrocellulose? 766 00:36:28,490 --> 00:36:31,417 You do this in undergraduate class, don't you? 767 00:36:31,417 --> 00:36:32,375 You don't do a western? 768 00:36:35,320 --> 00:36:36,112 We used to do-- 769 00:36:36,112 --> 00:36:37,820 AUDIENCE: Did it once in undergrad class. 770 00:36:37,820 --> 00:36:40,310 JOANNE STUBBE: Yeah, in what kind of a membrane? 771 00:36:40,310 --> 00:36:41,180 Was it in biology? 772 00:36:41,180 --> 00:36:42,757 AUDIENCE: Yes, biology. 773 00:36:42,757 --> 00:36:44,090 JOANNE STUBBE: So what membrane? 774 00:36:44,090 --> 00:36:47,650 Do you remember what the membrane was? 775 00:36:47,650 --> 00:36:50,015 AUDIENCE: I think it was-- it was not nitrocellulose. 776 00:36:50,015 --> 00:36:51,640 JOANNE STUBBE: It's not nitrocellulose. 777 00:36:51,640 --> 00:36:56,860 So this PVDF, polyvinyl difluoride is the standard one 778 00:36:56,860 --> 00:36:57,767 that people use now. 779 00:36:57,767 --> 00:37:00,100 It works much better than nitrocellulose-- this paper is 780 00:37:00,100 --> 00:37:03,010 really old, and so they're looking at nitrocellulose. 781 00:37:03,010 --> 00:37:06,980 So then they do this. 782 00:37:06,980 --> 00:37:10,960 And then, what do they do next? 783 00:37:10,960 --> 00:37:12,550 They have an antibody-- 784 00:37:12,550 --> 00:37:14,740 we'll look at the details of this in a minute-- 785 00:37:14,740 --> 00:37:16,600 that can recognize the protein, that 786 00:37:16,600 --> 00:37:19,540 can find it on the membrane. 787 00:37:19,540 --> 00:37:21,520 And then what we're going to see is-- 788 00:37:21,520 --> 00:37:23,300 you still can't see anything really, 789 00:37:23,300 --> 00:37:25,620 because you don't have very much material there. 790 00:37:25,620 --> 00:37:27,100 And you can't observe-- 791 00:37:27,100 --> 00:37:29,640 you don't have enough to stain, oftentimes, by Coomassie, 792 00:37:29,640 --> 00:37:31,670 so you're going to have to amplify the signal. 793 00:37:31,670 --> 00:37:34,930 So then you're going to make an antibody to an antibody. 794 00:37:34,930 --> 00:37:36,710 And then you have to figure out how to, 795 00:37:36,710 --> 00:37:38,590 then, amplify the signal. 796 00:37:38,590 --> 00:37:41,170 And we'll look at that in a second. 797 00:37:41,170 --> 00:37:44,260 Is this what-- you ran a western, is 798 00:37:44,260 --> 00:37:45,990 this what westerns look like? 799 00:37:45,990 --> 00:37:48,490 AUDIENCE: I remember, we first [INAUDIBLE] 800 00:37:48,490 --> 00:37:50,710 non-specific proteins to occupy the sites. 801 00:37:50,710 --> 00:37:52,210 JOANNE STUBBE: Yeah, so that's good, 802 00:37:52,210 --> 00:37:55,540 you have to block everything, if you're using crude extract. 803 00:37:55,540 --> 00:38:00,610 So in this case, we would be using the crude mixture-- 804 00:38:00,610 --> 00:38:02,110 well, not a crude mixture, it's been 805 00:38:02,110 --> 00:38:05,641 fractured by the ultracentrifugation that's 806 00:38:05,641 --> 00:38:06,390 been fractionated. 807 00:38:06,390 --> 00:38:09,290 But you still have mixtures of proteins in there. 808 00:38:09,290 --> 00:38:14,240 Have any of you ever looked at westerns in a paper? 809 00:38:14,240 --> 00:38:18,460 Or even the papers you had to read? 810 00:38:18,460 --> 00:38:20,290 The paper on the PC-- 811 00:38:20,290 --> 00:38:23,460 go look at the PCK-- 812 00:38:23,460 --> 00:38:26,890 PCSK9 paper, that had westerns in it. 813 00:38:26,890 --> 00:38:27,630 What do you see? 814 00:38:27,630 --> 00:38:33,170 Do people show you something that looks like this? 815 00:38:33,170 --> 00:38:36,839 And if they did show you that, what would it look like? 816 00:38:36,839 --> 00:38:39,130 So you have an antibody that's specific for the protein 817 00:38:39,130 --> 00:38:42,814 of interest, whatever that is-- supposedly specific. 818 00:38:42,814 --> 00:38:43,480 What do you see? 819 00:38:47,220 --> 00:38:48,350 What do you think you see? 820 00:38:48,350 --> 00:38:49,891 Do you think antibodies are specific? 821 00:38:52,410 --> 00:38:54,740 I think I have an example of a typical western. 822 00:38:54,740 --> 00:38:57,877 AUDIENCE: I don't think they're as specific as [INAUDIBLE] 823 00:38:57,877 --> 00:38:58,710 JOANNE STUBBE: Yeah. 824 00:38:58,710 --> 00:38:59,210 Yeah. 825 00:38:59,210 --> 00:39:03,669 So when you look at a paper, you should pay attention 826 00:39:03,669 --> 00:39:05,960 to this when you read a paper, if you're doing anything 827 00:39:05,960 --> 00:39:07,126 in biology, what do you see? 828 00:39:07,126 --> 00:39:09,680 You never see a gel, ever. 829 00:39:09,680 --> 00:39:17,339 What you see is a slice of a gel where they cut off 830 00:39:17,339 --> 00:39:19,880 this-- the way they cut up all this stuff and all this stuff. 831 00:39:19,880 --> 00:39:23,000 The reason they do that is because it's a hell of a mess. 832 00:39:23,000 --> 00:39:25,610 So let me just show you a typical-- 833 00:39:25,610 --> 00:39:27,920 I don't care what kind of an antibody 834 00:39:27,920 --> 00:39:30,740 you're using, in crude extracts, it's a mess. 835 00:39:30,740 --> 00:39:33,710 Because you have non-specific interactions. 836 00:39:33,710 --> 00:39:36,045 We'll just look at that. 837 00:39:36,045 --> 00:39:38,420 So that would be something like you might see-- depending 838 00:39:38,420 --> 00:39:40,290 on how much antibody you have. 839 00:39:40,290 --> 00:39:43,220 So when you see this, the reason everybody 840 00:39:43,220 --> 00:39:45,220 reports data like that now. 841 00:39:45,220 --> 00:39:49,130 So it looks like it's really clean, but in reality-- 842 00:39:49,130 --> 00:39:53,040 I think if it is dirty as that, then in my opinion, 843 00:39:53,040 --> 00:39:55,550 I would make you publish the whole gel. 844 00:39:55,550 --> 00:39:56,660 But people don't do that. 845 00:39:56,660 --> 00:39:59,840 They just cut off the little band 846 00:39:59,840 --> 00:40:02,840 they're interested in-- they can see it change in concentration 847 00:40:02,840 --> 00:40:05,097 using this method. 848 00:40:05,097 --> 00:40:07,680 But you should be aware of the fact that antibodies in general 849 00:40:07,680 --> 00:40:09,960 aren't as specific as you think they're going to be. 850 00:40:09,960 --> 00:40:11,206 Yeah? 851 00:40:11,206 --> 00:40:14,767 AUDIENCE: Are they required to report the whole gel in 852 00:40:14,767 --> 00:40:15,350 supplementals? 853 00:40:15,350 --> 00:40:17,686 JOANNE STUBBE: I mean, I think, it probably 854 00:40:17,686 --> 00:40:19,310 depends on the journal, and it probably 855 00:40:19,310 --> 00:40:20,730 depends on the reviewer. 856 00:40:20,730 --> 00:40:23,990 But I would say, we're going away from data-- 857 00:40:23,990 --> 00:40:27,700 is something that is a pet peeve for me. 858 00:40:27,700 --> 00:40:30,110 And all the data, which I think is all right, 859 00:40:30,110 --> 00:40:32,390 is published in supplementary information, 860 00:40:32,390 --> 00:40:33,390 as opposed to the paper. 861 00:40:33,390 --> 00:40:35,480 I think if you have something really dirty, 862 00:40:35,480 --> 00:40:38,680 you should publish in the paper, in the main body of the paper. 863 00:40:38,680 --> 00:40:40,430 If you have something that's really clean, 864 00:40:40,430 --> 00:40:42,179 and it looks like that, it's fine with me. 865 00:40:42,179 --> 00:40:43,790 You don't even have to publish it, 866 00:40:43,790 --> 00:40:46,880 if you could believe what people were saying. 867 00:40:46,880 --> 00:40:49,370 Because people know what this looks like, 868 00:40:49,370 --> 00:40:51,380 a lot of people-- everybody uses westerns. 869 00:40:51,380 --> 00:40:53,690 But if it's a real mess, then you 870 00:40:53,690 --> 00:40:58,070 need to let your reader know that this is not 871 00:40:58,070 --> 00:41:00,380 such an easy experiment, and it's not so clear-cut. 872 00:41:00,380 --> 00:41:03,500 That's what your objective is, is to show people 873 00:41:03,500 --> 00:41:06,650 the data from which you drew your conclusions. 874 00:41:06,650 --> 00:41:08,600 And then they can draw their own conclusions, 875 00:41:08,600 --> 00:41:09,870 which may be different. 876 00:41:12,410 --> 00:41:15,080 So let's look at the apparatus to do this. 877 00:41:15,080 --> 00:41:19,590 So how do you get from here to here? 878 00:41:19,590 --> 00:41:22,880 So you have a gel, you run the gel, a polyacrylamide gel-- 879 00:41:22,880 --> 00:41:23,950 what do you do? 880 00:41:23,950 --> 00:41:25,610 AUDIENCE: Put the membrane on the gel. 881 00:41:25,610 --> 00:41:27,000 JOANNE STUBBE: So you put the membrane on the gel. 882 00:41:27,000 --> 00:41:27,791 And what do you do? 883 00:41:32,995 --> 00:41:35,030 AUDIENCE: [INAUDIBLE] applying charges to. 884 00:41:35,030 --> 00:41:38,210 JOANNE STUBBE: Yeah, so you're transferring it 885 00:41:38,210 --> 00:41:42,420 based on applying a voltage across this system. 886 00:41:42,420 --> 00:41:45,570 So here's your gel. 887 00:41:45,570 --> 00:41:51,980 And here's your membrane, nitrocellulose membrane. 888 00:41:51,980 --> 00:41:56,000 And then they have filter paper above the gel, 889 00:41:56,000 --> 00:41:57,230 and below the membrane. 890 00:41:57,230 --> 00:42:01,040 Why do you think they have the filter paper there? 891 00:42:01,040 --> 00:42:03,455 When you ran the gel, did you have filter paper? 892 00:42:03,455 --> 00:42:04,090 AUDIENCE: Yes. 893 00:42:04,090 --> 00:42:05,046 JOANNE STUBBE: Yeah. 894 00:42:09,830 --> 00:42:12,280 How do you think they decide how to do this transfer? 895 00:42:12,280 --> 00:42:16,220 Do you think is a straightforward? 896 00:42:16,220 --> 00:42:19,470 Do you run it for an hour, do you run it for five hours, 897 00:42:19,470 --> 00:42:22,020 do you run it for 15 minutes? 898 00:42:22,020 --> 00:42:24,440 What is the voltage you use to do the transfer? 899 00:42:24,440 --> 00:42:27,620 Do you think any of that is hard to figure out? 900 00:42:27,620 --> 00:42:29,930 So how do you figure that out? 901 00:42:29,930 --> 00:42:33,820 Somebody told you that this is a good way to do it? 902 00:42:33,820 --> 00:42:36,410 Yeah, so that might be a place you start. 903 00:42:36,410 --> 00:42:39,500 So you do it because somebody gave you a recipe. 904 00:42:39,500 --> 00:42:42,230 But then what do you need to do to make 905 00:42:42,230 --> 00:42:46,604 sure this recipe is correct? 906 00:42:46,604 --> 00:42:48,020 AUDIENCE: Find out what conditions 907 00:42:48,020 --> 00:42:50,060 that work for what you're working on. 908 00:42:50,060 --> 00:42:52,520 JOANNE STUBBE: Right, and then how do you do that? 909 00:42:52,520 --> 00:42:56,620 So that's true, every protein is going to be different. 910 00:42:56,620 --> 00:42:58,886 And if you have a protein-- 911 00:42:58,886 --> 00:43:03,800 if you have a clean protein, versus a mess of proteins, 912 00:43:03,800 --> 00:43:06,410 and you try to do this transfer, the transfer conditions 913 00:43:06,410 --> 00:43:08,070 will be different. 914 00:43:08,070 --> 00:43:10,220 So for example, if you really want 915 00:43:10,220 --> 00:43:13,480 to look at the concentration of something inside the cell, 916 00:43:13,480 --> 00:43:16,220 in the crude extracts, you never compare it 917 00:43:16,220 --> 00:43:18,710 to a standard with clean protein, 918 00:43:18,710 --> 00:43:21,790 because this transfer is different. 919 00:43:21,790 --> 00:43:23,720 So you need-- in the back of your mind, 920 00:43:23,720 --> 00:43:25,910 if you care about quantitating this, 921 00:43:25,910 --> 00:43:29,090 you need to understand the basis of the transfer. 922 00:43:29,090 --> 00:43:34,920 So why do you think they have these filter papers here? 923 00:43:34,920 --> 00:43:36,830 So this goes back to what controls 924 00:43:36,830 --> 00:43:42,890 you would do to see whether your transfer was working. 925 00:43:42,890 --> 00:43:44,120 So what would you look for? 926 00:43:49,630 --> 00:43:51,660 Did you do this? 927 00:43:51,660 --> 00:43:54,110 What did you do? 928 00:43:54,110 --> 00:43:56,716 What did you do with the filter papers in your-- 929 00:43:56,716 --> 00:44:01,526 AUDIENCE: You want to filter all to the SDS molecules... 930 00:44:01,526 --> 00:44:02,692 JOANNE STUBBE: You did what? 931 00:44:02,692 --> 00:44:04,110 AUDIENCE: You want to filter all-- 932 00:44:04,110 --> 00:44:05,960 JOANNE STUBBE: No, that's not what you do. 933 00:44:05,960 --> 00:44:07,710 I mean, you might want to do some of that, 934 00:44:07,710 --> 00:44:11,370 too, but in terms of thinking about whether your transfer is 935 00:44:11,370 --> 00:44:13,030 successful-- 936 00:44:13,030 --> 00:44:16,860 figuring out the conditions to blot from the gel 937 00:44:16,860 --> 00:44:20,740 to a piece of paper is not trivial. 938 00:44:20,740 --> 00:44:24,660 And there is a standard way that you do this, initially, to try. 939 00:44:24,660 --> 00:44:27,180 But then you have to make sure that that method is working. 940 00:44:27,180 --> 00:44:28,746 And lots of times it doesn't work. 941 00:44:28,746 --> 00:44:30,870 So it's something that's going to be experimentally 942 00:44:30,870 --> 00:44:32,340 determined. 943 00:44:32,340 --> 00:44:35,640 So the question is, what would you 944 00:44:35,640 --> 00:44:38,880 think would happen if you did this for six or seven hours? 945 00:44:38,880 --> 00:44:43,450 Whereas, a normal blot would take two hours? 946 00:44:43,450 --> 00:44:45,730 AUDIENCE: Would be transferred onto the filter paper? 947 00:44:45,730 --> 00:44:47,760 JOANNE STUBBE: Right, it would go right into the filter paper, 948 00:44:47,760 --> 00:44:49,150 or even off the filter paper. 949 00:44:49,150 --> 00:44:51,150 So what you do is you take the filter paper out, 950 00:44:51,150 --> 00:44:54,360 you look for protein being bound. 951 00:44:54,360 --> 00:44:56,880 What about the gel? 952 00:44:56,880 --> 00:45:01,016 What do you do with the gel after your experiment's over? 953 00:45:01,016 --> 00:45:03,457 AUDIENCE: Make sure a protein's not on it? 954 00:45:03,457 --> 00:45:06,040 JOANNE STUBBE: Right, make sure that the protein is not on it. 955 00:45:06,040 --> 00:45:07,780 So these are simple controls, but these 956 00:45:07,780 --> 00:45:10,180 are the controls you always do until you work out 957 00:45:10,180 --> 00:45:12,590 the conditions to make sure this works. 958 00:45:12,590 --> 00:45:16,390 And it's pretty critical to make sure you have good transfer. 959 00:45:16,390 --> 00:45:21,730 So then, so this is the antibody thing that they do. 960 00:45:21,730 --> 00:45:23,920 Has anybody thought about these kinds of assays? 961 00:45:23,920 --> 00:45:26,170 You've seen them, I think, already in class. 962 00:45:26,170 --> 00:45:28,677 But what's wrong with this picture? 963 00:45:28,677 --> 00:45:30,760 The target protein, what's wrong with this picture 964 00:45:30,760 --> 00:45:31,400 in the target? 965 00:45:31,400 --> 00:45:34,300 So here's your nitrocellulose filter paper. 966 00:45:34,300 --> 00:45:36,040 What's wrong with this cartoon? 967 00:45:39,950 --> 00:45:41,400 Should be unfolded, yeah. 968 00:45:41,400 --> 00:45:44,130 So you're doing SDS page, it's unfolded. 969 00:45:44,130 --> 00:45:46,260 So then we react it with an antibody. 970 00:45:46,260 --> 00:45:48,210 Presumably we have a good antibody, 971 00:45:48,210 --> 00:45:50,490 but you've already learned in the first half 972 00:45:50,490 --> 00:45:53,520 of this course that having really good antibodies is not 973 00:45:53,520 --> 00:45:54,270 so trivial-- 974 00:45:54,270 --> 00:45:57,750 you can get them, but most of the time 975 00:45:57,750 --> 00:46:01,680 they are not specific if you're looking at crude extracts. 976 00:46:01,680 --> 00:46:03,630 They have little epitopes they recognize, 977 00:46:03,630 --> 00:46:05,490 if you're using monoclonals that could 978 00:46:05,490 --> 00:46:08,860 be present in other proteins. 979 00:46:08,860 --> 00:46:14,730 And furthermore, how are you detecting something? 980 00:46:14,730 --> 00:46:17,910 An antibody as a protein, it has absorption of 280. 981 00:46:17,910 --> 00:46:22,140 Again, this is too low to see, so putting an antibody on it 982 00:46:22,140 --> 00:46:24,216 is still going to be too low to detect. 983 00:46:24,216 --> 00:46:25,590 So how do you detect your signal? 984 00:46:28,440 --> 00:46:30,130 So have you done this? 985 00:46:30,130 --> 00:46:32,830 I'm surprised they don't do this in your introductory class-- 986 00:46:32,830 --> 00:46:35,380 they don't do westerns, at all. 987 00:46:35,380 --> 00:46:40,210 So what you're looking at is an antibody to an antibody. 988 00:46:40,210 --> 00:46:41,770 So you put your antibody on, that's 989 00:46:41,770 --> 00:46:43,280 specific for your protein. 990 00:46:43,280 --> 00:46:46,150 And then you make an antibody in another organism 991 00:46:46,150 --> 00:46:51,040 that can specifically recognize antibodies in general. 992 00:46:51,040 --> 00:46:55,940 So if this is to a mouse, you make it to go and isolate that. 993 00:46:55,940 --> 00:47:00,070 And then what you do is derivatize the second antibody 994 00:47:00,070 --> 00:47:00,700 with what? 995 00:47:00,700 --> 00:47:02,740 A protein? 996 00:47:02,740 --> 00:47:06,322 That can function as a catalyst. 997 00:47:06,322 --> 00:47:08,780 AUDIENCE: Why can't you just derivatize the first antibody? 998 00:47:08,780 --> 00:47:11,752 JOANNE STUBBE: Well, what? 999 00:47:11,752 --> 00:47:12,460 What did you say? 1000 00:47:12,460 --> 00:47:13,930 AUDIENCE: It's more expensive? 1001 00:47:13,930 --> 00:47:16,013 JOANNE STUBBE: Well, no, I don't know whether it's 1002 00:47:16,013 --> 00:47:17,760 more expensive or not. 1003 00:47:17,760 --> 00:47:19,062 But-- 1004 00:47:19,062 --> 00:47:21,330 AUDIENCE: Well, because you'd have to derivatize 1005 00:47:21,330 --> 00:47:22,290 every primary antibody. 1006 00:47:22,290 --> 00:47:24,331 JOANNE STUBBE: So you'd have the derivatize every 1007 00:47:24,331 --> 00:47:27,100 primary antibody, and so this is a standard procedure. 1008 00:47:27,100 --> 00:47:30,080 You could derivatize the primary antibody. 1009 00:47:30,080 --> 00:47:32,716 So that's not a bad question. 1010 00:47:32,716 --> 00:47:34,090 And so what you're doing now, you 1011 00:47:34,090 --> 00:47:38,890 can buy these commercially, so they have rabbit, rabbit, 1012 00:47:38,890 --> 00:47:40,930 mouse, whatever, antibodies. 1013 00:47:40,930 --> 00:47:44,230 And the key is the amplification of the signal, 1014 00:47:44,230 --> 00:47:48,370 and you use enzymes to amplify the signal. 1015 00:47:48,370 --> 00:47:50,620 Does anybody know what the enzymes 1016 00:47:50,620 --> 00:47:54,348 are, what the enzymes do to amplify the signal? 1017 00:47:54,348 --> 00:48:00,569 AUDIENCE: You can covert the molecule to a blue molecule... 1018 00:48:00,569 --> 00:48:02,360 JOANNE STUBBE: To something that's colored. 1019 00:48:02,360 --> 00:48:05,780 So does anybody know what that horseradish peroxidase-- 1020 00:48:05,780 --> 00:48:08,360 have you ever heard of horseradish peroxidase? 1021 00:48:08,360 --> 00:48:10,750 So that's a heme iron-- we're going 1022 00:48:10,750 --> 00:48:12,650 to be talking about heme irons pretty soon, 1023 00:48:12,650 --> 00:48:14,210 and hydrogen peroxide. 1024 00:48:14,210 --> 00:48:17,000 It makes a chemically very reactive iron oxide 1025 00:48:17,000 --> 00:48:22,190 species, that can oxidize a dye that changes color. 1026 00:48:22,190 --> 00:48:24,300 And it has extremely high extinction coefficients. 1027 00:48:24,300 --> 00:48:26,819 So you can see it, and it does it catalytically 1028 00:48:26,819 --> 00:48:28,610 and the lifetime of the dye is long enough. 1029 00:48:28,610 --> 00:48:30,650 So it accumulates, and you can get really 1030 00:48:30,650 --> 00:48:32,030 amplification of your signal. 1031 00:48:32,030 --> 00:48:35,780 Or you can use a phosphatase that 1032 00:48:35,780 --> 00:48:38,840 liberates something that's highly colored, again, 1033 00:48:38,840 --> 00:48:39,870 and you can see it. 1034 00:48:39,870 --> 00:48:45,180 So this is a standard method that everybody uses. 1035 00:48:45,180 --> 00:48:48,050 And so, that's our gel. 1036 00:48:48,050 --> 00:48:51,380 So now we're looking at sort of-- 1037 00:48:51,380 --> 00:48:53,990 at the end already-- but we're looking at these gels, 1038 00:48:53,990 --> 00:48:56,580 and what do you see through the different steps? 1039 00:48:56,580 --> 00:49:00,830 So if we look through the first gradient, 1040 00:49:00,830 --> 00:49:05,890 through the sucrose gradient, that gets us through DNE. 1041 00:49:05,890 --> 00:49:10,580 And if you look, say, at lane E-- 1042 00:49:10,580 --> 00:49:14,990 our goal is to separate proteins that 1043 00:49:14,990 --> 00:49:18,450 are specifically localized in each one of these membranes. 1044 00:49:18,450 --> 00:49:20,810 So you need to believe that's true, 1045 00:49:20,810 --> 00:49:24,470 that people have selected the right group of proteins 1046 00:49:24,470 --> 00:49:25,120 to look for. 1047 00:49:25,120 --> 00:49:27,974 And you notice they do more than one. 1048 00:49:27,974 --> 00:49:29,390 So they look at multiple proteins. 1049 00:49:29,390 --> 00:49:31,090 Why do you think-- 1050 00:49:31,090 --> 00:49:35,770 do you think it's easy to select the proteins to look for? 1051 00:49:35,770 --> 00:49:38,220 And why or why not? 1052 00:49:38,220 --> 00:49:41,240 So, they obviously have selected a group of proteins, 1053 00:49:41,240 --> 00:49:44,240 and I think most people would agree that they've selected 1054 00:49:44,240 --> 00:49:46,160 a good group of proteins. 1055 00:49:46,160 --> 00:49:48,980 But what do we know now about proteins, 1056 00:49:48,980 --> 00:49:51,706 do they stay in one place? 1057 00:49:51,706 --> 00:49:55,760 No, they move around. 1058 00:49:55,760 --> 00:49:58,250 But some might be present in very low amounts, 1059 00:49:58,250 --> 00:49:59,960 sometimes in much higher amounts. 1060 00:49:59,960 --> 00:50:04,340 And so you need to have more than one protein as a control 1061 00:50:04,340 --> 00:50:06,800 to make sure you're looking in the right region. 1062 00:50:06,800 --> 00:50:09,500 And what do you see in E? 1063 00:50:09,500 --> 00:50:12,900 If you look over here, it tells you what the organelle is. 1064 00:50:12,900 --> 00:50:16,400 And if you look at this protein, this 1065 00:50:16,400 --> 00:50:19,880 is localized to the lisosomes-- we talked about that in class. 1066 00:50:19,880 --> 00:50:22,010 If you looked at this protein, it's 1067 00:50:22,010 --> 00:50:24,080 localized to the peroxisomes. 1068 00:50:24,080 --> 00:50:29,360 So in addition to the ones we care about, the ER proteins, 1069 00:50:29,360 --> 00:50:31,730 we're also getting proteins that are 1070 00:50:31,730 --> 00:50:34,010 localized in other membranes. 1071 00:50:34,010 --> 00:50:39,320 So that's when they went to the next method, 1072 00:50:39,320 --> 00:50:42,200 and they added on another gradient 1073 00:50:42,200 --> 00:50:46,290 to try to separate out, again, the lysosomal 1074 00:50:46,290 --> 00:50:49,900 and the peroxisomal proteins. 1075 00:50:49,900 --> 00:50:52,740 And you can see they were pretty successful at this. 1076 00:50:52,740 --> 00:50:56,180 There's none of these proteins left in this gradient. 1077 00:50:56,180 --> 00:50:58,370 So that's good. 1078 00:50:58,370 --> 00:50:59,790 And they took it a step further. 1079 00:50:59,790 --> 00:51:01,250 Do you remember what this is? 1080 00:51:01,250 --> 00:51:04,202 What are they looking for down here, in this? 1081 00:51:04,202 --> 00:51:05,410 AUDIENCE: Enzymatic activity. 1082 00:51:05,410 --> 00:51:07,160 JOANNE STUBBE: Yeah, so enzymatic activity 1083 00:51:07,160 --> 00:51:08,840 is localized in certain organelles. 1084 00:51:08,840 --> 00:51:11,090 So they again did a second experiment 1085 00:51:11,090 --> 00:51:12,720 to look at all of that. 1086 00:51:12,720 --> 00:51:14,950 So they were very careful in this, 1087 00:51:14,950 --> 00:51:16,340 they figured out how to separate. 1088 00:51:16,340 --> 00:51:17,780 And that's the key thing for them 1089 00:51:17,780 --> 00:51:22,220 to analyzing the concentration of cholesterol 1090 00:51:22,220 --> 00:51:23,180 in these membranes. 1091 00:51:23,180 --> 00:51:25,130 And what they looked at-- we're over time-- 1092 00:51:25,130 --> 00:51:27,980 but is the concentration of cholesterol compared 1093 00:51:27,980 --> 00:51:30,570 to the total amount of lipids. 1094 00:51:30,570 --> 00:51:34,220 And how did they do that analysis? 1095 00:51:34,220 --> 00:51:36,860 Gene Kennedy, who's at Harvard Medical School-- 1096 00:51:36,860 --> 00:51:39,530 he's in his 90s, now-- really trained all the lipid 1097 00:51:39,530 --> 00:51:42,250 chemists in the whole country. 1098 00:51:42,250 --> 00:51:44,000 And they figured out many years ago 1099 00:51:44,000 --> 00:51:47,187 how to separate lipid fractions with methanol, chloroform 1100 00:51:47,187 --> 00:51:49,520 extract, something that you guys probably haven't though 1101 00:51:49,520 --> 00:51:51,140 about at all. 1102 00:51:51,140 --> 00:51:53,870 But we're really pretty good at separating things, 1103 00:51:53,870 --> 00:51:56,090 and it's nothing more than an extraction 1104 00:51:56,090 --> 00:52:00,410 like you do as organic chemist to purify and separate things. 1105 00:52:00,410 --> 00:52:01,740 We've figured that out. 1106 00:52:01,740 --> 00:52:07,220 And so then they use mass spec to allow them to quantitate 1107 00:52:07,220 --> 00:52:09,680 the amount of glycerol. 1108 00:52:09,680 --> 00:52:16,730 And then in the end, so they use mass spec, these western blots, 1109 00:52:16,730 --> 00:52:20,510 and they can change the concentration 1110 00:52:20,510 --> 00:52:23,420 of the cholesterol and do the experiments over and over 1111 00:52:23,420 --> 00:52:26,660 again, to see what happens. 1112 00:52:26,660 --> 00:52:32,180 And when they do that, this is the picture of cyclodextrin. 1113 00:52:32,180 --> 00:52:35,060 So you can see the only difference is this group here 1114 00:52:35,060 --> 00:52:38,020 versus that with a methyl. 1115 00:52:38,020 --> 00:52:42,050 And one, so this is hydroxypropryl-- 1116 00:52:42,050 --> 00:52:47,000 hydroxypropionyl cyclodextrin-- so 1117 00:52:47,000 --> 00:52:50,240 it's like a cavity like this. 1118 00:52:50,240 --> 00:52:53,330 And the other only other change here is a methyl group, 1119 00:52:53,330 --> 00:52:54,500 removing that. 1120 00:52:54,500 --> 00:52:57,230 And they have very different properties 1121 00:52:57,230 --> 00:53:00,560 about binding and releasing cholesterol, which somebody 1122 00:53:00,560 --> 00:53:04,310 had to do a lot of studying on to be able to ensure that they 1123 00:53:04,310 --> 00:53:07,520 can use it to remove cholesterol, 1124 00:53:07,520 --> 00:53:09,860 and then to add it back to the media. 1125 00:53:09,860 --> 00:53:12,154 And so you have to think about the exchange kinetics, 1126 00:53:12,154 --> 00:53:13,820 you have to think about a lot of things. 1127 00:53:13,820 --> 00:53:16,760 This is not trivial to set this up, 1128 00:53:16,760 --> 00:53:21,070 to figure out how to control the levels of cholesterol. 1129 00:53:21,070 --> 00:53:24,560 And then what they do is, this is like a typical assay, 1130 00:53:24,560 --> 00:53:26,480 and this is the end. 1131 00:53:26,480 --> 00:53:29,960 What you can do is this, removes cholesterol, 1132 00:53:29,960 --> 00:53:32,300 and you can see it change. 1133 00:53:32,300 --> 00:53:37,940 This reports on low levels of cholesterol, 1134 00:53:37,940 --> 00:53:41,090 which is happening over here, allows the protein 1135 00:53:41,090 --> 00:53:43,820 to move to the nucleus where it's smaller. 1136 00:53:43,820 --> 00:53:46,460 And that's how they do the correlation-- the correlation 1137 00:53:46,460 --> 00:53:48,110 between the levels in the nucleus 1138 00:53:48,110 --> 00:53:50,850 and the levels of cholesterol. 1139 00:53:50,850 --> 00:53:53,960 So I thought this was a pretty cool paper. 1140 00:53:53,960 --> 00:53:56,630 And these kinds of methods, I think, 1141 00:53:56,630 --> 00:53:59,370 will be applicable to a wide range of things 1142 00:53:59,370 --> 00:54:03,800 if people ever do biochemistry, looking 1143 00:54:03,800 --> 00:54:05,470 at the function of membranes. 1144 00:54:05,470 --> 00:54:08,620 So, OK, guys.