Speaker 1: In this video, we are going to cover the core content you need to know about blotting for the MCAT. Since the MCAT cares most about the interpretation of blots, we will spend most of our time focused on that and will only cover the method of preparing a blot briefly. In general, a blot has three major steps, running a sample on a gel separated by size, transferring the samples from the gel via blotting, basically, pasting a piece of special paper on top of the gel to absorb the samples, and finally, visualizing the sample using an antibody or a couple of antibodies designed to detect a specific molecule or set of molecules. For example, let's say you wanted to know how much adenosine diphosphate and adenosine triphosphate was present in three different cell line cytosol. You would first extract the cytosol from those cells, run them on a gel, transfer that gel to a blotting paper, then expose the blotting paper to a marker designed to detect both ATP and ADP, then look at the blot. Since the samples were run on a gel first, we should expect to see larger molecules showing up near the top of the blot. For example, in this blot, the ATP marker should be above the ADP marker, since ATP is bigger than ADP. Additionally, the thickness of the bands can also differ from one another, where thicker bands represent more of whatever molecule is being identified. For example, cell C contains the largest amount of ATP in its cytosol out of all of the cells since it has the thickest band in the ATP marker, while cell A contains more ATP than ADP, which is in contrast to cell B, which has more cytosolic ADP compared to ATP. Remember, the major takeaway from all of this is that the location up and down in the gel tells you about the size of the molecule, with larger molecules appearing near the top of the gel and smaller molecules appearing near the bottom, while the thickness of the bands tells you about how much of a particular molecule is present, with thicker bands indicating more of a particular molecule and thinner bands representing less of a particular molecule. Now that we have a general understanding of how gels work, let's answer a question on this topic. Here this question asks, truncated titin, a protein responsible for the elasticity of muscle fibers, is being investigated for its role in causing dilated cardiomyopathy, DCM. It is hypothesized that individuals with higher amounts of truncated titin are at greater risk of developing DCM. If this hypothesis is true, which of the following patients would be at the highest risk of developing DCM based on the following western blot? To start, let's break this question stem down a bit. Here we are told that truncated titin, or a shorter version of titin in high amounts, is thought to lead to DCM. Then we are asked to find the patient who has the highest risk of developing DCM. Said another way, we need to find the patient with the largest amount of truncated titin. To do this, we will need to interpret the blot we have been given, which according to the caption, shows both truncated and normal versions of titin. First, let's figure out which part of this blot shows truncated titin. Since truncated titin would be shorter than regular titin, and thereby smaller, we should expect it to show up further down in the blot, since smaller molecules will end up traveling further in the gel, prior to being transferred over to the blotting paper, so we will only focus on the second band in this blot. From there, we need to determine which patient has the most truncated titin. To do this, we will look at the band size. Remember, the thicker the band, the larger the amount of stuff being measured in that sample. So in this case, patient C has the most truncated titin, while patient D has the least. This means patient C has the highest chance of developing DCM, and therefore, answer choice C must be correct. Now let's discuss the three specific blots the AAMC expects you to know. These include southern blots, northern blots, and western blots. Outside of some minor methodological differences between the blots, they all work the same way, except they measure different things. Southern blots measure DNA, northern blots measure RNA, and western blots measure proteins. We can remember which goes with which by using the mnemonic snowdrop. In this mnemonic, we stack both words vertically on top of one another. Since the S aligns with D, we can remember that a southern blot measures DNA, and since the N aligns with R, a northern blot would measure RNA. And since the W aligns with P, we can remember that a western blot measures proteins. Here the O can be thought of as a zero, since it is simply a placeholder to make the mnemonic work. By measuring RNA and proteins, scientists are also able to measure how transcription and translation are affected by different things. Specifically, transcription, the creation of RNA from a DNA template, can be monitored by a northern blot, while translation, the creation of a protein from an RNA template, can be monitored by a western blot. If translation is downregulated, we would see normal amounts of RNA, but the western blot would show low protein, indicative of something interfering with the process of translation. However, if transcription was downregulated, we would see low levels of RNA and low levels of protein, since protein production is being stopped at the level of transcription. Let's look at a couple of questions to see how the MCAT could test us on these concepts. First up, this question asks, which of the following could be used to determine whether or not someone has HIV by testing for anti-HIV antibodies? To answer this question, we need to first determine what it is that we are measuring. In this case, it is anti-HIV antibodies. Antibodies are proteins, so we will need a technique that can identify proteins. Using Snowdrop, we can see that the western blot corresponds with proteins, making C the correct answer here. The other answer choices are wrong because they measure either DNA or RNA. Specifically, southern blots measure DNA, while both northern blots and RT-PCR measure RNA. Although we haven't talked about RT-PCR, you can see it as having the same function as a northern blot, they simply differ in how they measure the RNA. Now let's look at another question. Scientists noticed that a population of frogs lacked limbs when exposed to agricultural pesticides. They hypothesized that the pesticides blocked sonic hedgehog protein production at the level of transcription. To verify this, they should use a northern blot or a western blot, that's the first part of our 2x2. And will that thing measure the amount of protein or the amount of transcripts produced? That's the second half of the 2x2 question here. To answer this question, we will again start with what we are measuring. In this case, it is transcription or the process of converting DNA to RNA. This means that we will need to measure the level of RNA in these frogs to see if transcription is being blocked. Again, using the snowdrop mnemonic, we can see here that we will need a northern blot to do this, therefore answer choice B must be correct, since it correctly identifies the type of blot and what that blot can measure, here RNA aka transcripts. Answers A and D are incorrect on the basis of content alone, since they mismatch northern blot as measuring protein and western blot as measuring RNA. Finally, answer choice C is incorrect because measuring the amount of protein here would tell us whether or not translation was being affected, but the question asks us to pick the technique that would look at transcription instead. In summary, we learned that blots are used to measure the amount of different molecules in samples. Larger molecules will end up closer to the start of the blot, while smaller molecules will end closer to the bottom of the blot. The larger the band on a blot, the greater the amount of that molecule in the sample. There are three specific blots we need to know for the MCAT. These include southern blots which measure DNA, northern blots which measure RNA, and western blots which measure proteins. We can remember which is which by using the mnemonic SNODROP, keeping in mind that the O doesn't stand for anything. In addition to measuring RNA, northern blots can tell us about transcription, while western blots can tell us about the level of translation in a cell by measuring the protein levels present. If you enjoyed this video and found it helpful, make sure to like and subscribe for more helpful MCAT tips, and share it with anybody else who's taking the test.