Again, so welcome back everyone that's watching on Moodle or later on Moodle, since I'm not allowed to stream onto platforms at the same time because of Amazon rules. But let's just continue with the lecture. And all right, so talk about a little bit of more of bioinformatics, right? So if we are dealing in proteins in bioinformatics, then of course we are interested in the protein structure, because the structure of the protein gives it its function. So there are a lot of computational tools available. I think many of us did it. You all reported the guy. I think that one report or two reports is enough. Like Twitch is running a very aggressive campaign against bots watching, and not only that, but also selling followers or buying followers. That's just like app. You just get your followers by being funny and giving lectures, right? That's how I do it. Or at least I try to be funny. You don't have to think I'm funny. Anyway, so structure prediction tools, there are a lot of different structure prediction tools, but they fall into these kind of categories. So the Appinico prediction is kind of the unsolved question in bioinformatics. So how do you take the sequence of amino acids and then predict the tertiary or quaternary structure from it? Because that's the thing that we want to know. Primary and secondary structure are not too hard for computers. And so the secondary structure prediction is more or less solved. There's a lot of tools out there online. We will actually do a secondary structure prediction, or I will do one live for you guys. And I think in the assignments there's also one. And then transmembrane helix prediction is also kind of solved. It works relatively well. So if you have an alpha helix and this alpha helix has hydrophobic side chains, then this will preferentially be in the cell membrane. So what you want to know about a protein is, is this protein in the cytosol of a cell? Or is it in the cell membrane? Or is it, for example, in the membrane of the nucleus or in the endoplasmatic reticulum? And these things, so the structures making these things able to be in the cell wall, they are pretty well known. Because the cell wall has a certain thickness. So if an alpha helix has a certain amount of helical revelations, or how would you call it? Helical turns. So if there's a certain amount of helical turns, then you can kind of combine that with the knowledge of how thick the cell membrane is. And then you can predict if this protein will insert into the cell membrane and if it will function, for example, as a, as a, as a transporter or as a receptor. Then there's something called threading or fold recognition. This is based partially on homology. But threading and fold recognition is its own prediction method. And then there's homology modeling. And homology modeling is the way that you use known structures from other proteins to predict structures in new proteins. So if you know that if I have a sequence of amino acids, which is 50 amino acids, and I know that these 50 amino acids have a certain sequence in protein A, which has been solved by x-ray crystallography, then you can then use this known structure to say, well, OK, so in this protein, I find 44 out of 50 amino acids. So it will be very similar to the one that we already know. All right, so this is, as so, the Appiniccio prediction is an algorithmic process by which protein tertiary structure is predicted from its primary sequence. And this is still a remaining unsolved question. And it is one of these top 125 outstanding issues in modern science. So at a certain point, I think in 2010, they made a list of which things are solved in science and which things are outstanding, so which things are not solved yet. And the idea is that if you would solve this question, so if you would come up with a program and you would feed this program the amino acid sequence and it then predicts the tertiary structure, then this would be like a massive advancement in science. And it actually would be, because we would be able to much better be able to deal with what is the function of this protein and how, for example, can we target drugs or other interventions to kind of block this protein or to make this protein work better. So there are some projects out there which try to do this. And one of the most interesting projects that I find is Folding at Home. So Folding at Home is based on the SETI at Home program, so the search for extraterrestrial life. So it uses your home computer. Or you can have this program run on your home computer. It communicates with a server. The server is located at Berkeley. And it gets little data packages from Berkeley. And then it does the analysis. So it does local, it tries to locally fold the protein as best as possible and then send this back. So it's kind of a crowdsourced project. One of the other projects which is very interesting is the Folded Project. So the Folded Project is actually something that you can play on your smartphone or on your tablet. And it's a game. So in the game, you are the one folding protein chains. And if things are hitting each other, you score negative points. And if things are more or less free from each other, then you get a positive point. So you get a score for how well you fold the protein. And data from this project has actually led to some very novel algorithms. Because apparently gamers are much better at folding proteins than scientists are. Because scientists have all these preconceived notions on how a protein should look like. But gamers don't really have that. So using this game and using strategies that gamers came up with, they developed novel scientific algorithms. And there's a couple of nice publications on the Folded Program. So you can just Google it and you could download it and help. Because hey, it's just using human intelligence to kind of brute force this project. And then there's the Human Proteome Folding Project. And the Human Proteome Folding Project is more or less a scientific project run by different universities to kind of solve this open issue in modern science. All right. So when we talk about secondary structure prediction, this is very well done. Cheers. Yes, have a nice holiday. So the secondary structure prediction is kind of solved. So we can solve secondary structures with around 80% accuracy, which is more than good enough. So hey, there's programs out there like RaptorX, SS8, or SimPredict, or YASP. And these are programs which you feed the primary amino acid sequence. And it will then predict the local secondary structure. And it does this relatively well. So hey, it will detect if there's an alpha helix or a beta sheet and some other known protein structures, which are secondary structures. These are structures based on the atomic bindings and based on the hydrogen binding, so hydrogen bridging. All right. So this is then used to predict transmembrane helixes. And these programs fall into more or less four different categories. So there are programs like HMM Top, which use hidden Markov models. You have MMSOT, which uses neural networks, so kind of machine learning. And then you have Phobius, which uses homology prediction. So hey, it uses the homology of known proteins to predict novel proteins. And then you have CC Top, which is using constrained consensus topology. So this is kind of a mixture between hidden Markov and homology prediction. So it uses homology from head. So that's the constrained consensus. So the constrained consensus comes from the known proteins. And then the topology part is using hidden Markov models to kind of fold the parts that which have no homology to known proteins. So I wanted to show you an example. So I just put some links here. So I'm going to switch you guys over to my Firefox window.  So what is your favorite gene or your favorite protein in human or in mice or in plants? And then we can just search for that. So let me go actually to my window. So of course, we are going to search in the protein database. So if you guys have a favorite protein, let me make this window a little bit bigger actually so that we have the search button here as well. It will squeeze a little bit so I can make it a little bit bigger like this. All right, so otherwise we will just do the insulin receptor, which has some nice structure, right? It's a receptor tau protein. That's probably one of these that is like these unstructured proteins in Alzheimer, if I remember correctly, right? But we can do the tau protein. So we just search for tau protein. So let's see how CCTOP will go there. So this is the partial. This is a fragment. So this is also partial, partial, partial. Let's sort by not the default order, but just by, can I not search by the length of this? There should be an, well, let's not make it too big, right? Because if we make it too big, then the prediction will take like a couple of hours, and we can't see it. So let's just take one of the first ones. So this is 124. I have fleeing, don't know what a krkota, krkota is. I always like to figure out which animal we are dealing with then. So Spotted Hyena. So who would have guessed that a Spotted Hyena has such a nice scientific name? Krokuta, krokuta.  Like, we really got shafted on the scientific name, in a way. But so let's take the tau protein, partial sequence, 860 amino acids, which is quite big, so it might take a while to do the prediction, from the Spotted Hyena. So it's loading the sequence, so it gives us all kinds of information, right? And it tells us what the CDS is. But here, this is the thing that we want. This is the protein sequence. So we can't use it directly like this. We have to export the sequence. And of course, we are going to do this in FASTA format. At least we don't have the name Huso Huso, like the Meluga Sturgeon. I don't know. I like the double name, like Pissang Pissang. It's nicer. But all right, so we just take the FASTA, right? So some of them are actually really, really interesting. I actually have a list of my favorite scientific namings of animals. Anyway, so when you have the FASTA sequence, we can just copy-paste it. So I'm just going to select it, press Control C on my keyboard. And then I'm going to go to CC Top, which I already opened. So CC Top, and like you can see, the program is very easy. You just give it the primary sequence. So we just copy-paste this in. You can fill in your email if you want to not wait for it. But we're just going to submit, and we're just going to wait here a little bit, so we can just talk about it. And I'm already going to click here. So we have to wait a little bit. And in the meantime, we can actually open up the results that I did before. So I can just copy this link or not. Just click it. Yeah, so I can just click the link. So this is the insulin receptor in humans that I did before. So this actually shows, of course, the insulin receptor. So here you see that it actually predicted that part of this alpha helix will be embedded in the cell wall, because this is kind of its way of showing that this might go through the cell wall. And then you see that it has an unordered amino acid or an unordered part of the chain. Then at the end here, there's another little alpha helix. So this is kind of what it predicted. And here in the legend, it says that blue is outside, is the exocytosolic, the yellow part is in the membrane, and the red part is inside of the cell. So this part is inside of the cell. This part is outside of the cell. And then we have the re-entrant loop, which is not there. And we don't have any green, so we don't have any interfacial helices. Of course, we can also look at the summary. So this was the insulin receptor in humans. It actually figured out that this thing just has known structures. Then we can go to the 1D structure. So that's just a standard structure. And now it colored it to say which parts are inside, which parts are in the membrane. And then we have here this little part, which is here in the 3D structure. So it's, I think, this part here. No, this part here. So it's part of the helix, which it doesn't really know if it's inside or outside of the membrane. And then here you have the inside of the membrane again. So then it will predict the 2D structure as well. So here it shows you the 2D structure prediction. And it will run different prediction algorithms. It will run the standard HMM top. But it will also look at other. And then it will combine everything. And then I'm zooming out, which I don't want. I would just want to scroll down. And so here it will have all of these different signals. Oh, I don't want to zoom. I want to scroll down. So here it says that there are all kinds of different signals. And these things you can get more information on by looking at it. And it will do a prediction on these are generally known proteins in which it finds the same sequence. But the nicest thing is that you can just click on the 3D structure, and then it will tell you more or less how it looks. All right, so did the CC top already finish? So we just have to refresh it? No, it's still being processed. So we will come back to that later. Hey, you can see that it's just a very easy program. You just throw in the sequence, and then it will do a prediction. And it will show you if it's inside or outside. And hey, you get some more 2D information about the 2D structure. And unfortunately, you can't really click on it. But here you have the cross refs. So these are all other proteins which have homology with your sequence. And then it shows you which part of the sequence it has homology with. So some of them are really weird because there's only a very small homology. But some of them have like large homology. So this 2DTE-TGE has the same kind of membrane helix as that we see in the human insulin receptor. All right, so let's see. Let's refresh again. No, it's still running. So we could have taken a smaller one, but let's just continue with the presentation. And I will click refresh. And when it's finished, then we can see what it does with the tau protein. All right, so let's go back to the presentation. So I just put this in. And this is more or less the permanent link if I want to go to the results. Because it took some time. Like when I ran it this morning, it took around 15 to 20 minutes to go through all of the predictions and then combine them into one figure in the end. All right, so there's two other methods, which of course had a CC top uses several methods. So the ThreadFold recognition is you can use when there is no homologous protein sequence available. It's template-based. It's similar to doing local alignment to known protein. So what it does, it actually takes the whole protein sequence that you have, then chops it up into little pieces, and then looks if any of these little pieces is available in another protein. And this is, of course, very similar to homology modeling. But in homology modeling, it's more like doing global alignment. So if there is, it just looks at the whole protein sequence, looks if there is a protein sequence of similar length, which has a known structure. And then it just does the modeling based on that. And then had the changes of the novel protein towards the known protein, it will then try to do the prediction of that. So of course, homology modeling is much quicker. Because it takes a known structure and then just makes modifications to the known structure while in the ThreadFold recognition, it chops up the protein into little pieces. And then for each of these little pieces, it looks to see if there's a known structure available and then combines them together into one big novel structure. So if you can do a homology modeling because there's a homologous protein out there, then you can kind of do that. All right, so the latest and greatest. So this is the new slide that I added for you guys. You can still see that I didn't format it. But this is Google. So Google actually thought it would be a good idea to get involved as well, because they bought one of these. Well, it's not a supercomputer. They bought one of these quantum computers. So from the D-Wave. So they bought this 512 qubit D-Wave machine. They didn't really know what they wanted to do with it. But this machine is one of the first real quantum computers in the world, which you can just buy. So the D-Wave company is located in Canada. And Google bought one of these machines. And last time when I was at Oak Ridge National Laboratory in the US, I actually asked them, do you have a D-Wave? And they said, no, we don't have a D-Wave, but the guys in the building next to us, which is the kind of NSA building. Well, not NSA, but Department of Defense building. They might have one, but we can't go in there because you're not a US national. So you're not allowed to enter the building. So then I was really disappointed because I really wanted to see one of these new computers, but they didn't have one at that time. But what Google did is they combined two of these things. So they have a neural network, which does their secondary structure prediction. So it predicts all phalluses. Happy holidays for you. Yes, happy holidays for you as well. Yeah, on the? Yeah. So you have to stop with the wrong one. That's OK. So yeah, just wishing colleagues happy. Next week I will already be on holiday. I have to see if we actually have a lecture next week. If we have a lecture next week, then I'm probably legally obliged to show up and give you a lecture. But we can talk about that. But so what Google did is it took this neural network, which is used in a lot of different prediction algorithms for predicting alpha helices and beta sheets. And then the next step that they did is so head. This is the first step. So they take the protein sequence, and then they have these databases. These databases feed this big neural network that they build. The neural network then spits out a distance prediction. So this is a prediction of how far away individual amino acids are from each other. And then besides that, they have this angle prediction. So that's the angle of the side chain, because of course the side chain cannot kind of fold back 180 degrees. So this is kind of a constrained matrix, which they have. So if there are very abrupt changes in the side chain, then that is generally not how it works in the real world. In the real world, things gradually loop around. But what they then did is then they used this new big D-wave quantum computer of theirs. And then they did gradient descent. So gradient descent is an algorithm in which you start kind of wiggling stuff around. And at every position of the wiggle, so you move one amino acid a little bit, and then you calculate the overall scoring of the protein. So in this case, they're looking at free energy. So you calculate the free energy and see if the free energy goes down. So if the free energy goes down, you say, oh, this was a good wiggle. And if the free energy goes up, then this is a bad wiggle. Because in the end, everything in the universe wants to be at the lowest energy state. So this is how they wiggle their protein. And they published this in January 2020. And this actually predicts tertiary structures. And it does it pretty well. So on average, they have like a 75% accuracy, which is really good. So they are on track to kind of solving this open question in science. So very good. Like applause for Google. It's not just a search engine, but the company itself is like massive. And you can read more here on the DeepMind article. If you're not really into reading like these articles that are written by the authors themselves, you can actually go to the Nature publication that they have. I think it's still available for free. So you don't have to pay until like the end of the year. Because it's such an important article, or it's deemed such an important article on how they do it. So they kind of combine these two methods. So the one method is just using this neural network to do standard kind of homology prediction in a way. And then they follow this up by gradient descent in which they start wiggling the amino acids. And then every time calculate the free energy of the whole structure, which of course is a very computationally intensive methodology. So you would require like a massive computer farm for that. Of course, Google has a massive computer farm. But on the other hand, they also have this D-Wave machine. And this D-Wave machine is optimized. So it's a quantum computer which does gradient descent, which runs a gradient descent algorithm much, much faster than a standard computer can. Very interesting work. So just read the article and if you're interested. And it's going forward in science. It's always good to see that things are improving. And like 10 years ago, this was completely unthinkable. That you would be able to predict a protein tertiary structure with like this level of accuracy. All right, so production of proteins. We already talked about this a lot. So I actually cut down the number of slides talking about the ribosome again. But I do want to highlight some of the features of the ribosome. So like proteins are assembled from amino acids using information encoded in gene. So we already talked about that. And then the genetic code is a set of three nucleotides each, right? So three nucleotides on the DNA in the coding region. They code for a single amino acid. So this is kind of the description of that. We know that DNA contains four nucleotides. So now a question to you guys. If we know that there are four nucleotides and that there are three, a combination of three nucleotides code for a single amino acid, then how many possible codons are there? How many possible amino acids can you encode on DNA? 64, could you write that down in a more scientific way? Like how did you get to the answer? Right, we learned about how to write things down in R. So if you would write this down in R, how would you do this? Right, because it's something to the power of something. All right, 4 to the power of 3. Very good, very good. You guys are getting really good at this. Should come up with more difficult questions for during the lecture and making sure that you're still paying attention. But yeah, in theory, there's 64 possible codons. There's 20 amino acids. So why is that? So there is redundancy. There has to be redundancy, because if you have 64, because the DNA can code for 64 possible amino acids, and in humans, there are around 20 which are essential and there are 20 which are coded by tRNA. So there is some redundancy in the genetic code. So genes are encoded into DNA, transcribed into mRNA, pre-MRNA, modified into mature, and then the ribosome is making these. So HE is known as translation. Proteins are always biosynthesized from N-terminus to C-terminus. That's why we write them down in that way. So ribosome picture, we already saw this. HE, the A-side, P-side, and E-side. It's all in the RNA lecture. And here, like indeed, four to the power of three means that we have 64 possible codons. So there are 60 cents codons for amino acids because there are three terminator codons because you need to tell the ribosome to stop at a certain point. And there is one codon which is used as the initiator, because you have to tell the ribosome also to start making a certain thing. And so some of the codons are redundant or degenerate, as we call them. So two or more different codons are coding for the same amino acids. And so we know that tRNAs are the things that match the mRNA codon. So how does it end up that there are only, like there are actually 60, so your body actually makes 60 tRNAs, but some of these tRNAs, they come with different anti-code. So the total number of tRNAs found in the system is less than 61. And in most cases, it's only 22 to 31. So even if you look at the most complex plant, it only has 31 unique tRNAs. So in the 1960s, people were already aware of this. And the thing that FC Crick from Watson and Crick proposed is that the anti-code on 5 prime end in the tRNA has the ability to pair with more than one base found at the third base pair at the 3 prime end of the codon, so of the mRNA. Because the orientation of codons and anti-codons is anti-parallel, so they go the other way, the third base of the codon base pair with the first base pair of the anti-codon. So how does this look? So if we have Lloyd's scene, right? So this is twice a tRNA, which codes for Lloyd's scene. Here, we see that the codon, which is on this tRNA, is G-A-C. Well, actually, it's not. It's G-A-G. So we read it from 5 prime to 3 prime, right? And here, we see the mRNA, which is written from 5 prime to 3 prime. So it is C-U-C. So this is the perfect match. And then the tRNA has a perfect binding to the mRNA. However, there's also the other Lloyd's scene codon, which has the same sequence. But this is actually bindings to C-U-U. And how can it bind to C-U-U? Because normally, the G would not be able to bind to U. But if you look into the ribosome, then the wobble base pair position is actually not really read very accurately. Because the tRNA is not horizontal. And it's not at 180 degrees or at a 90 degree angle. It's actually slightly at an angle, which is slightly less than 90 degrees. So that means that the third base pair, like this G here, together with this U, they are not that close together. They are kind of in a wobble position. So there's a little bit of slippery there. So the third base pair on the mRNA for each of the codons does not really matter that much. And that is because the tRNA is at an angle, which is not exactly 90 degrees. But it is slightly at a less angle, which makes that the third kind of base pair in the codon does not really need to be correct. All right, so that was the only thing that I wanted to kind of add to the ribosomal stuff. I don't think that we mentioned the wobble bases before, but the wobble bases are really important because they can show you if something is species-specific. So it's just a nice analysis method, too. Because the third base pair is also not really under selection. Normally, all of the base pairs in the genome, which code for proteins, they are under selection. Because if you change the amino acid coding of the protein, then that has an influence on the biochemistry. But in this case, the third base pair is not really under selection. So you see more mutations at the third base pair location than you see at the other ones. All right, let me actually refresh the tau protein. So the tau protein worked really well because, let me show you guys, there are no 3D structures for this protein. So boohoo. Although we can still learn something right, because we can still see that there are some which are homologous. So you can actually see that there's a domain at the end of the tau protein. And this domain is PF00418. So we can just Google that. So PF00418. So PF00418. So that's a tubulin binding. So there is a tubulin binding thing. So it teaches us that at the end of the tau protein, this is the wrong one. So here, how we learned that at around amino acid 600, up until 720 of this tau protein, we see that there is a structure which is very similar to a tubulin binding thing. And if you want to learn more, then it is actually something that it has to do with microtubule assembly. So that's the microtubule within the cell. So that's interesting. So did it find anything else? What we can learn from? So there are prior tau proteins. And there is also something called PS00. And that's the same thing. So it actually doesn't find that much structure here. So apparently, people haven't really studied tau proteins, or the structure of tau proteins. One of the reasons for this could be that they are really hard to crystallize. If you can't crystallize them, then it's also really hard to get a structure for them. But that's interesting, that it doesn't find any 3D structure. So it's not the best example then. I'm actually glad that I ran the insulin thing before. Instead of trusting you guys to come up with one of the few proteins which actually have no 3D structure yet. But it does give you the 1D structure. It gives you the 2D structure. Sorry. No, don't be sorry. That's how these things work. Like, in the end, you want to learn things about proteins. So you're looking at the protein structure. And here it also gives you some ideas that there might be some other things, like these are the predictions that it does. But it doesn't really contain a membrane part. But there is a very, very well-known domain which runs from amino acid 600 to 720. Philip? Yep. Keys. There you go. Very good. All right. So, tau protein. So it's finished. So if anyone has its other or its own favorite protein and thinks, look, oh, that's something that we should just have a quick look at, then sure, throw it in the chat. And we can just restart the prediction and update the prediction counter with one. So that's OK. All right. So talking more about protein. So proteins are, I hope now that you guys get a little bit of an understanding that proteins are kind of complex, that they are difficult to predict in some ways. But if you're lucky, then someone made a 3D structure of your protein somewhere. So then that helps you to understand what your protein might be doing. So of course, when we talk about proteins, we have to talk about how you can identify proteins, right? Because we need to know which protein is which. And for that, we need to start first with the purification. Because if you have a cell and you break open a cell, then literally 200,000 different proteins come out. And then you need to find a way to kind of get your protein of interest. So there are different purification techniques like ultracentrification, precipitation, electrophoresis, and chromatography. Just say it of Dutch when it doesn't work in English. Chromatography, chromatography, chromatography. And there are different identification techniques like immunohistochemically using x-ray crystallography or NMR or mass spectrometry. And of course, we can use two-dimensional gel electrophoresis. But we will just quickly go through all of them because what is ultracentrification? So ultracentrification is a step where you do centrifugation, where you separate certain organelles from the cell for further analysis, right? So the first thing that you do is you take your tube, you put your cells in there, and you put some lysis in there so you break the membrane open. And then when you break the membrane open and everything starts floating around, like here you have in different colors and with different sizes, you have different proteins. So what you then start doing is you do repeated centrifugation. So you have a nice centrifuge, you start spinning stuff around, and then the bigger proteins start falling to the bottom, right, because they are heavier. So if you spin at a certain speed, then the heavier ones will more easily drop to the bottom than the lighter ones. And at a certain point, hey, you have a little pellet, and what you do is you take the supernatant and then you bring it into a new tube. This new tube is being spun again, some other proteins, hey, so you spin it at a higher revolution, others drop to the bottom, and so you continue until you have your protein of interest, right? Of course, at each of these steps, you can remove the supernatant and you could also study the green ones, the red ones, the blue ones, or the really, really small, tiny black ones, right? Because at each of these steps, you can kind of stop. And the nice thing is, is by spinning it at a certain amount of revolutions, you also have an idea of how big the protein is going to be, which is going to go out of solution into the little pellet on the bottom, right?  So that is why in the old days, when we were talking about proteins, proteins would be mentioned by their Dalton number, because this is one of the first techniques. So they would identify proteins and they would say, oh, this protein is 100 Dalton, meaning that it's like 100 size units. And if it would be 40 Dalton, it would be like, well, four out of 10 from the bigger one, right? So all of the classification used to be based on sizes. So sizes or weights, more or less weight, which is coupled to size, of course, in a way. So one of the other things is precipitation. So when you do it with centrifuge, then it's called sedimentation, but what you do is you have a solution, then you add something to the solution, and then, for example, you add a salt to it or nitrogen, or nitrogen-nathrium or chlorine, and then you put that in, and then this binds to the protein of interest, and then that falls down, and then you have something called precipitate, and you have the supernatant, and then you can study the precipitate, or you can study the supernate. But hey, if you have something which binds, and not all things will bind to all proteins, then you can very precisely extract some of them. Electrophoresis is the nowadays most common technique. It's still very much used in the lab, and here in electrophoresis, you use the motion of dispersed particles relative to a fluence under the inference of a uniform field. So for DNA and RNA, you do electrophoresis in one dimension, but for proteins, you do it in two dimensions. It was developed in 1807 already, the electrophoresis, and so what you do is you make a gel. This gel has kind of holes in there, so things can travel to the holes, and because DNA and RNA and proteins are charged, like they have a slightly negative charge or a slightly positive charge, you can pull them using an electrical field, and then because you pull them through an electrical field, they get slowed down by things like friction, because they bump up to the little kind of matrix that is in the gel, and then they get slowed down. So the bigger a protein is, the more it gets slowed down, so if you pull proteins through a gel, then the big ones will remain on top, and the small ones will kind of run easier, so they will go down to the bottom much more. So this holds for DNA and RNA, but of course this also holds for proteins, but for proteins you generally want to separate them into two dimensions, so you want to separate them based on the isoelectric point, because I told you that proteins have amino acids which have positively or negatively charged side chains, and so the pH at which a particular molecule carries no net electrical charge is in the statistical mean, is the isoelectric point. So because every protein has a different number of charged side chains, or so positively or negatively charged, each protein, when you put it in water, will change the pH of water to be higher or lower than seven, or it will remain seven if the thing is uncharged. But what you do is if you put proteins in water, then based on the side change, you will have a different pH, so a different net charge, so what someone then figured out is that you can actually separate proteins in this way. So what you see here is a gel, so in this gel there is a pH gradient from being very acidic to being very basic, right? So and because of this gradient in the X direction, proteins, when you put them on the gel, will start moving from left to right and start finding the point at which they are not charged because that is the point where they want to be. So if I have a protein which normally is basic, it will start moving until it reaches, so if the protein in water has a pH of eight, then it will start moving and at a pH of six, then the whole thing will be neutral, so it will start moving and that's what you see in this direction. Besides that, you're just using the standard gel electrophoresis where you pull them down based on the size. So here on the bottom, we will have the small proteins, here on the top we will have the large proteins and then here they are separated by their isoelectric point, so that means that that proteins which normally have a positive charge will go to the left side, positives would have a negative charge will go to the other side and the more charged they have, the more they will move. So you see very clearly here that there are spots and you can see that from this gel, you can see many, many different spots and you could use like a pipette or a needle to extract proteins very carefully from this gel and every spot here is a single more or less protein, so it might still be a little bit of a mixture but hey, you can see that you can very, very clearly separate like up to like 200 to 500 proteins from each other which is much better than what we could do before using centrifugation. Using centrifugation, it gets really hard to separate 200 different proteins from each other, right? You always end up with a mixture because everything just drops down and you just take the whole thing. So this is a major advantage in protein study because now here we are able to pick individual protein and we can also quantify them because the size of the dot is the amount of protein that was there. So if you would do this on, for example, the extract of a normal cell versus the extract of a cancer cell, then you might be able to see that, well, in a normal cell, this is a very little dot but in a cancer cell, this is a huge dot, right? That means that the cancer cell is producing much more protein than the normal cell is producing and that might give you a clue of how you would be able to treat this. Then there's another nice way of separating proteins which is based on chromatography. So chromatography is something that you might have done as a child.  There are many different mixtures, many different chemicals in this, so you put a little dot on the paper, you put it in a bucket of water, so something like this and you need a little piece of paper like this, which you then put into the water and then what happens is, is that because of suction tech or because of suction, right?  So the mixture is dissolved in a fluid, so you would put this thing in the water, you would put the thing into the thing and then it flows through another material, which is the stationary phase. So the paper is the stationary phase and in this case, the mobile phase would be water. You could, of course, also use things like whiteboard cleaner or acetone or whatever, right? So, and then had the various constituents of the mixture travel at different speeds and this causes them to separate. So if you would take like a green marker, someone like this and you would do this using a little cup of water and a little piece of paper, and then you would see that there are different, different, that every constituent, so every element of the green marker travels at a different speed. So you would get a picture like this and of course this holds for proteins as well. You can just put your protein mixture, take a little piece of paper, put the little piece of paper in and then the water, together with the proteins will run up through the paper and then this will separate out the different proteins as well. So it's also a way that you could separate proteins from each other. All right, so that was a quick overview. If you wanna purify your proteins, you can use centrifugation, you can use precipitation using things like salts. You could use electrophoresis, which is the standard way nowadays of doing it, but you could also use chromatography. And of course, there's not a lot of bioinformatics involved in all of this, but that's because we need to be able to separate proteins before we can start using bioinformatics to analyze them. Of course, in 2D GL electrophoresis nowadays, there's a lot of bioinformatics involved because I have had projects where I got pictures like this, so a couple of five, 600 of them, and then the question was, can we use these pictures and have a little program that actually recognizes the spots, aligns the pictures, right? Because every gel will look a little bit different because of the pH being not exactly the same and the charge not being the same. And so this little program that you can write will pick a couple of control spots and then for each of the pictures, it will make them similar, and then it will do an analysis of how much of a certain protein is there. So 2D gel electrophoresis, there are tools available which allow you to load in like 100 of these gels, and then for each of the gel, it will give you a list of proteins, so it will use like the pH on the one axis, so it will tell you that not the name of the protein, but it will say protein with a PI of 8.7 and a size of 300 or a size of 100 is expressed one in the first gel, 1.5 in the second gel and 1.3 in the other gel. So there's a lot of recognition going on. So you have picture recognition, then making sure that all the pictures are lined up, and that works actually very well and it's actually very fun to do to see that. Our computer can actually help you analyze data coming from this. Same thing here for chromatography. If you have like a professional chromatographer in your lab, then also there, there will be a lot of bioinformatics involved because bioinformatics will do kind of the band detection, band picking, and also will kind of see if the sizes of the bands or not the sizes, but the content of the band is similar or different between different gas chromatography runs that you do or water chromatographies that you do. All right, so I've been talking again for 48 minutes. Let me see. I still have around 20 slides left. So that means they were really, really nice on time. So I will take a short break again. So you guys can enjoy the second break. I think this one is cats. Do we already have an animated gift series on cats or not? I think we already had some animated gifts on cats, right? I don't know, but at least I got it. Yay cats, yeah, always cats, like cats on the internet. Not enough cats on the internet. Now there's nowhere near enough cats on the internet. All right, so I will take a little toilet break, do a little bit of a cigarette break and drink some water again. And I will be back in 10 to 10 minutes, something like that. I might be a little bit earlier. And of course, since I'm showing cats, there will probably be these bots that get attracted to the cats and want me to buy more followers, which we're not going to do. We're just gonna ban the bots and make sure that they don't spam the chat. All right, there's actually someone for dogs. For next week's Curita, I will make a special dog break for you. So I will find the animated gifts of dogs wearing silly outfits and stuff and make one of them for you. All right, so I will, yeah, no problem, no problem. I will stop the recording.


I'm gonna do some easier heroes first, um, I shouldn't go here, but yeah. Alright, day 7, so let's get down here. Yeah, I'd be probably, there's a square of movement wasted there I think. Gala, I said that I made a boo-boo with Gala and I can't remember what it was now. Oh yeah, you're already at the maximum, I thought you had expert, what have you got there? Basic intelligence, oh man. Yeah, he's not as amazing as I thought actually, I'm Gala.  Day 7, alright. I want to get the fire, I want to get the energy elementals because they're actually useful for something. Does that make me a bad person? Given the troops yet, so I can do everything I want to do. Um, if in doubt, do the thing you know you're gonna want to do. We've got tons of Mercury, this is affordable, and Phoenixes are incredible. And there she blows. Um, now just to hedge against week of plague, I might just buy these now. Um, it's not too bad, I don't want to have to make that decision beforehand. Alright, what is this guy gonna give me? Five grand. Pay what I promised, I never record you promising me five grand. And what am I gonna do? Say no? Hourglass, it just cancels luck. Like, I'll just take the five grand I guess. Disappointment. Considering how much rigmarole there was for me to get it to him. Five grand is an insult. Um, does he need to stay here? It doesn't matter that much. Try and do the Nagas. I can have an after, if you can get that on the way back. Okay. This guy, why not? I'm still waiting for my comeuppance here. I want a fantastic hero to come galloping out of here and take the conflicts back. Let's find out. We're letting some mullet go. Hello? See, weird stuff can happen at this stage of the game. What? Dima's just maybe stumbled upon me, realized he can't possibly beat me. So he thought he'd gallop on. Yeah, let's just get this fight done. He has 24 spell points.  Look at him. He's a baby hero. Let's just see what he does. What have you got? What are you doing? Yeah, there you go. Meteor Shower's decent. He's the Meteor Shower hero. That's his special skill. That's his kind of gimmick. Why is your maroll so trashy? You've got one, two, three, four troop alignments. Why doesn't the AI care about that? I don't know. I do nothing. Why didn't the Meteor Shower hurt the dwarves, I wonder? Maybe they had the resistance trigger. Try not to lose any troops and minimize the amount of spell points I'm earning. Just wait. He's gonna go again. Good. Okay. I should be able to raise all that back. Yes, it does feel good. Resurrecting grand elves. Feels right. I feel like I'm Tolkien or something. Or Iluvatar. He had no artifacts. Okay, cool. We do immediately take the shackles off again, okay? There's a case for viewing air here as well. There is a case for it, I think. I'm torn as to what to do now with her. Should I go underground? Should I go back up to here and explore some more of the green and... There's an acropolis down there at the other end of this one? Or a cover of darkness one or the other? I don't know. Let's get this guy moving. Do this. I think we've got some easy choices to make with all these heroes. Go here. Get this. Come back up. No point sandowners there, no. That's it. Then go here. Plan A. Go in here. Get these. And I need to be able to get to you, which I can. And he's gonna take on the Nagas. 31 grand goes very quickly. We've got four Phoenixes a week. It's so good. So now that we've invested in these, we're just gonna pile into them both. They're just so strong. These aren't going anywhere without an upgrade, so that's easy. Tomorrow? Here's a question. Is he gonna be able... Is Fiona gonna be able to go here, here, down to here, wherever the boat is in one day? Maybe not. So I'm gonna have to come up to meet him. Can I do this now? Nagas summon boat in one day? Maybe not. I might have wrecked... I might have messed up here. So if I'm taking energy elementals away tomorrow, give me these today. I think that's still okay against the Nagas. I might have overdone it there because Valeska needs money. She needs money for these. Okay. Okay. So with my 1500 bucks, probably buy all these. We're just letting them build up again, I think. These can kill the Nagas, right?  Does this thing have morale? Does it care about morale? It does. Phoenixes care about morale. Who knew? I won't need the harpies. I'll split them into two groups, maybe, for this fight. He's not gonna make it. Sloppy. Do I bother with the Nagas or do I come back later with energy elementals tomorrow? I guess I'll hedge now.  Don't need that. Right, we'll wait for tomorrow. We're going to get these. And I can actually wait till tomorrow to upgrade as well, because I don't have enough money. Right, none of that answers the question of what we're doing with Ibiza. So for now I have no clue where that is. Always worth having a look, because for one movement of tile, you get a big reveal. I think this is near an Acropolis. I think that we were up here before. It's the tan. I think that tan colored portal is up here. Right, let's do this and then we're planning to just town portal back. See what's down here. Oh, there is no cover of darkness to overwrite whatever. Okay, so maybe... Any nice skill? I do have a skill up my sleeve. I could chance it. She might teach me something useless. Hellhole. Which we have far along. I want to go here until the town portal back. And everything is ritually interconnected. Oh, I see what's up here. What if I keep following this path? Okay, that's that one there. It's an underground. I keep thinking I'm gonna find a town on the other end of it, but... This is valuable. These are valuable reveals, I suppose. Keep going. It's safe for me to do this, right? If I lose the conflict, I can just reconquer it. Oh my God. This is bizarre. You never see this. Big, long, straight path to nowhere there. So there's an inferno up here somewhere. Well, alright. My main mission is to get Veleska to get in a boat. And that still seems to turn away. Dragging Ents through the snow. Okay, well here she is. So we're gonna start upgrade buying these guys.  I've got 12k up my sleeve. These guys, as things stand, 8, 7. Can't buy them all. Can't do this at the same time. What's he gonna do for me? He's gonna get me some gold. I'm not gonna be able to get them all, I don't think. 10 grand well spent. These guys are quite good. Alright, here's your army. Now I'll go get this.  So she has speed 7. I'm speed 9. This is okay. Hopefully just kill them right? Yeah. So I think a better idea is to come over here with no retaliation. Let these waste their move. A bit of a morale risk, right? They didn't kill as many as I thought they would. Damage 15 to 25.  It's just okay, I don't love it. Okay, so no morale. No morale, don't be silly. You're not. You have bad rail. Good. And reach me. Guy's pear shaped. This should be a kill. And yeah. These guys no retaliation, movement 9. Can't cast spells on them. Gela does have a... Ice bolt up his sleeve. Could be time to cast a spell. Just to make sure of things. He's got no artifacts here. He's stuck on power 7. So that actually only kills 1. If that only kills 1, I'm gonna take... Damage and response. That's not what I want. It's very pinnickety, but... 10 SP just isn't that valuable compared to losing, say. Four arrow elementals or something or whatever. Whatever they lose. I don't know. Good reward. Very good reward. Very much worth it. Okay. Plan A. Get in a boat. Catch is... I don't think Fuhrer can help him with anything further. So... Yeah, this idea of the earth elementals and stuff. Just have to wait for some other time. Maybe Libby can come and pick them up for us later. Now, that 6 grand will help a lot. What are we doing here? We're buying this... We're buying this out? Yeah. I'm investing. Don't have enough, but maybe I could buy it all the time. Five grand each. What are they? Five grand each for a Titan. She's going to have the rampart creatures. She's going to have the tower. So that's Ents, Elves, Unicorns, Majors. Say Gremlins, Titans. It's 6. I could bring the gargoyles with just because they're excellent. Decide tomorrow. This isn't... as important as getting the Feleska moving, so we'll wait. He hasn't got much movement left anyway. And do we want to invest? I think we do. I think that's okay in terms of money. Okay. Blue's desperate. Sorry to hear that. I can't help but feel partially responsible. Why do they do that? Oh, you're so dead. You're cruel. It feels wrong. What am I doing after I kill them? I don't know. Who cares? Let's just get this done. We have to warp right now. Yeah, so they take extra damage. Yeah. They take extra damage from the lightning bolt. Vay is a decent hero. She might actually have some stuff we want to steal. So that's my weight. We're just going to be spamming Resurrect at this stage. I haven't seen any reason not to.  Nothing particularly scary. Come on down.  It's like one week of troops. I don't really want to have to cast Resurrect twice. But 10 Elementals is enough of a reason to... Just killing them now. Okay, we're back. Vay, don't you... I don't care if the Great Moon Bowls attack something.  Okay. Resurrect being put to the challenge here. Better not lose an L for you. I can't resurrect the Storm Elementals.  Do I want to spend... 16 spell points for an elf? Yes. It stinks. Can't resurrect Elementals. I found that out last time we played. Two days ago. That stinks. But, oh, landmine. These are terrible artifacts. Terrible. Okay, her last slot. Do we want air magic now? To eventually become an expert air mage. What do I know in the air that I wish I had? I don't know chain lightning. I don't know lightning bolt even. I just don't like being... I don't like having three schools of expertise. It just seems like... You can only cast one spell at a time. Man, I'm salty about the 21 Elementals. That stinks. Major salt. What's happening with the rest of the army? What's going on here? Can I have some stuff, please? Come down here. Do this. Okay, Gela's getting to the point where he's... going to want an army. Closest point to the red portal is here. Gee, that's inefficient, isn't it? He needs Water Walk. Need to learn Water Walk. She only has six heroes too, so I could always hire some more heroes. I don't have enough money to just buy the two Titans. I don't have enough money to send her off yet. She might have to wait the next day. Let's buy them there. She can't leave him as well, wait. She can leave. She could leave without them. Without... I mean without these other troops. The other troops I was thinking of buying is these. Which is 1200 bucks, pointless. 3520 for the obsidian gargoyles. It's done now, isn't it? Look at that. Just went and did it. Controversial. Yep, she's tired of waiting around. You know, watching her life is now. I bet you she can't summon a bird after this today. She can try. Tribe bit failed. Probably can only try one today. Tribe bit failed. How many times am I allowed to try? Burning through spell points. She burned half her points. But there is a little thingy here. So maybe I should go here before I... That's too much momentum lost. Is there any wishing wells up here? Anywhere in the... Teleport zone. This guy's here. Ooh, Nagash. Hello. Can I get to you? Several Chaos Hydras. Lots of mighty Gorgons. This is a proper hero. This might be their best hero. Let's have a look. Okay. I said before, I said a while ago, Orange is winning. This is not their best hero. Idiotically with a few skeletons accompanying this fantastic fortress army. Minus two to morale. Why the AI does that? Yeah. Anyway, so I can do some mean things to this with army, I think. He comes here. There's another one here. She's easy. She's up there in that fresh zone. Doesn't show me who he is. 13 vs. level. They're just going to have to be wasted. Ooh, the inefficiency. She does have a day of tourism. Up her sleeve. Don't want to fight Nagash. So how close is he to... There's an underground portal there. It's pretty far from there. Could it go? We could just continue up here. Have a look at this inferno. Come on. There it is there.  Unless she lightning bolts my air elementals, which would make me sad. So I could take this town and then actually it's another town for us to have take this town and come back up through this portal here to challenge this area again if we have to abandon it. Hire another hero out of here to explore the area. It's blocked off on this side, which is nice. Yeah, this feels okay. No need for the shackles, right? Don't need the bag of gold either. Looking to cast inferno. What does this do anyway? Knowledge by two. Not as good. Don't care about that. Landmine isn't as far on ever going to cast. Resurrection online. 30 of 30 is annoying. Let's see what she does with it. Oh! What? How do you salivate? Did they attack my dwarves? What happened there? What did the devils do? I wasn't watching. They attacked the elves. I'm not sure what to do. I don't even feel like I need to slow everyone. Doesn't really do much. Although they will be annoying. Seems kind of dumb. I thought that would work. Oh! Right, so if I do this... That hurts, right? The server, I would be careful with those things. Because they retaliate. Not in all directions, right? I know they don't retaliate because I already... Okay, okay. I like this move. But then did the familiars go first? Movement 8. I think maybe the familiars go first.  That doesn't matter. It's beneficial to do this. He's got speed 9. Actually, no, it tells me. These guys go before the familiars. But not before the server eye. So if I want the first turn, I can kill the server eye now. Or I can use my 23 to deal about let's say 200 damage. Which is about 50 of them. In fact, I'm tempted to resurrect. Yeah, I think it's expert slow here now. She could cure the archdevil, which would be atrocious, but never too late for expert slow. Okay, so let's change the order of everything I get to shoot. Yeah, I get to shoot everything. Don't even worry about it. I'm going to take care of everything. Just get it. Just get it. Don't even... This will kill one devil. I don't see any reason to wake them up though. Walk up here so the magogs can't do their thing.  Okay. You do nothing. In fact, have your turn. I actually don't know if I'll have enough in the tank to resurrect or anything. So is it resurrect now? Because you work on them. You're still blind. I'm going to resurrect now. It might have shown me how many HP it will resurrect. I really don't want to lose this many spell points and or troops to this fight, but that's what's happening. Alright, these guys are ready. Kill that stack. Kill either of these stacks. Which one's more? 4x100 and something. This one's harder to kill, right? Defense 8? Defense 20 though? I'll try. Okay, it mattered. Alright, come on down here. Don't want damage to me. Okay, so I obviously want to shoot the archdevils with these.  I did resurrect from one elf last fight. It was very decadent of me. Now wait, wait, and now resurrect. I forgot about the rest of the people I have to kill. That's going to be embarrassing. Yup, it is. Deeply embarrassing. Okay, so I'm going to be resurrecting. The towers go again, boom. Oh, a waste. What a waste. You're watching the most inefficient idiotic player in the history of the history of a waste of mana. Look at this. Oh, woeful. Unhappy. This town better be good. Something, no troops, but that's okay. Fully developed. Archdevils are here. What do these guys sell? I've never needed these. I'm glad running out of ammo isn't really a thing in the game. Thank goodness. Candent. So I could stay the night. Risk losing the game if I get trapped by Nagash or someone else. Don't really fancy that. Got 177. That's just a tragedy. The story of those fellows. You guys might as well stay here, just point this. In which case you guys might as well come back and do that. Welcome to Hell, dwarves. Oh, hey, mage guild. Not much to write home about. Lightning Bolt is here. Oh, Curse is here. I'm not going to bother with that. I'm not going to stay there. I'm just going to use her spell points to do this. I don't want to use town portal yet. I'll assume that's taken. I'd rather reveal a bit more map. Ooh, I want to get that. Observatory. I want to get this other stuff here. So that makes me want to buy a hero. Which I don't have the money to do. So... All over the shop, aren't I? It's red that's mucking around up there. Okay. Okay. I'm going to look at him. It's Tameka with the army. Okay. So I lingered. Well, I had to linger, though. We worked that out. I had to linger. I didn't want to fight this army. This is just... This is really boring to watch, but... Never mind. Did that seem strong to you guys? They're not why I've been in Monarchs. That's good. See, this is all valuable. She can't resurrect them. This is why you're leaving behind a basic force. Just pick your beefiest guys. Leave them behind. And you'll kill nine lizard men. Oh, you're so dead. Now she can't get back there quickly. Where did he go, Nagash? He... He went down here then. I'd love to kill this army. But she's going to have to do it the old-fashioned way. By galloping... hard on the plains to here. See if we can catch her. Because we can murder this army while it's attached to a hero that doesn't know what she's doing. Can we eat large hydras? Pretty nasty. But she had no spell points. She's a terrible hero. I can't get Labitha any support. I can't get her any... reinforcements. Come back. What are you doing? Nothing, maybe? I was going to spend money here. No, he's not. Okay. She's not going to get back in time. She's got a gallop all the way out to here. If she doesn't start galloping now. If she just goes portal to the rampart right now. Go get to the thing from the warlock. Maybe these green elves don't... I just don't bother galloping back to here to upgrade them. But I run out and get the unicorns. What am I getting from here? Is that it? Worth while? Worth the spell points? I reckon so. I reckon so. Could be idiotic. Let's find out. Get here. Get what we came for. Centaurs or anything like that. Get the four unicorns that are here.  Get these. You keep the green guys. Do I want to give you the artifacts while I'm here? Yeah, you could have the air and start casting it. Neither of us are air mages, so... Probably two tricky overthinking things, but whatever. Go back. Come down to here. Get ready to fight. Something tells me she could have got there. She could have actually galloped her way up to her inforter. No. No. Okay. Just misses. So a lot would have to go wrong here, right? Like N'Gar should have to come up here. You have to gallop up through the rough terrain. Get to the blue. Get the army off to Mika. She'd gallop up. Give it to him. He'd come under here. Which against a human might be... Darn it. She can't use that. Biddledee. We're going to come up this one, which isn't even in the right location. So we've got to come up here. What can I go this way? I can't go that way either. I can't even take a shortcut through there. I've got to go all the way around underneath here. Or I guess I can come up this way. To use it. It's another whole day off. Okay. Well, it's one more day. They get to consolidate their position. I'm not doing the Earth Elemental thing. I don't think we're doing that anymore. There's an extra day. I was going to say for Alaska maybe. That's not even very good. Oh god, I'm doing this. That's something to do with Alaska. It's not very exciting, but it's not insane. Those ideas go. One, two, three, four, five, six, seven. We go here. Shakti goes boom, boom. I need to run out. He's running out of moves.   I don't know why I did that.  Let's have a look. What are they going to do? Okay. They're very interested in the green. Wow, they've all disappeared back down to base.


Hi everyone, I'm Elayna and I'm your trainer for the next few minutes. How was your lunch? Awesome. Yeah, I'm very pleased with the lunch options here at War Camp Miami, so really cool. So how many of you want to have a blog or have a blog? Awesome. Well, we're going to be talking all about blogging and I want this to be a very interactive session. So I want you to ask any questions that you may have. I want you to tell me what your concerns are because my purpose here is not to go through the PowerPoint, it's to actually know that I contributed to your blogging journey. Is that okay? Yeah. Awesome. I mean, you're free to tweet and to Facebook and all of that. You can take pictures, so there's no limit on that. Before we get started, I want to give you a definition, let me see if this will work here. Apparently not. Okay. There we go. I want to give you a definition of blog and bolt because I wanted to use a very awesome title for this session. So I called it taking your blog from blog to bolt. And what I mean by blog is lacking action and who can read the last one? Hopeless. Yeah. And I don't really think that any blog whatsoever is hopeless. I think that we can feel hopeless because we don't know what the next step is. So hopefully this session will help you in that area. Now who can volunteer to read what bolt means? You? Okay. You're a beautiful lady here. Stand up so they can see how beautiful you are. Underbolt says business opportunities, reads, and generos. Generos. Woo! So this session is all about getting credibility, visibility, and profitability. If you are not making money with your blog, then you don't have a blog in business. You have a hobby. Okay? Business means business connections, business opportunities. It means people buying your products. It means getting media exposure. So we're going to be talking about all of those things that you can get out of blogging. My favorite thing that I get out of blogging is that I get to talk to the coolest moms on the planet and teach them about how I raise my kids and how they can raise theirs. Of course you may think like, wow, she's so bossy. But really it's not about that. It's really being an influencer. And I want each of you to think of yourself right now as an influencer, as a person that can make a difference because you have influence with your words.  Awesome. Now turn to your partner, to the people sitting next to you and tell them that the world is waiting for my words. Awesome. So that's the kind of energy that I wanted to generate in the room before we get started. Because really that's what this is all about. None of us are hopeless. We can all do this and as we do it, we can inspire other people. So those are all the things that you saw on the WordCamp Miami website that we're going to learn today. You can take a picture but I'm not going to read them for you. But we're going to learn a lot of stuff and if you don't learn something, then you can smack me in the head. I'll give you permission right now. Lightly and gently, okay. Awesome. You're laughing. So that means you don't want to take a nap right now. I was worried about that. Okay. So the first thing that we're going to touch on is the horrible, the common things, the common reasons why your blog may not be working. How many of you already have a blog in the room? Okay. So some of you may be feeling like I have this blog and it's thin, you know, it's kind of working but there's something missing. So I'm going to go through three things that may be the reason. The first one is that you are failing to have a strategy or a plan. As you see right here, it says fail to plan equals plan to fail.  I always said that web design happened was the very, very last step. Okay. Designing, coding and putting the pieces together, that is the last step of the process. The first step is to do a brain dump to really plan out what you're going to do. And you're probably saying, well, Alina, I don't know everything that I want to do and that's fine. You don't have to know all the pieces. Just the next pieces to get you started. And then says, you know, any strategy is a good strategy. There is no bad strategy. I always say that God rewards action. As you move, you're going to really understand if that's the right move or the wrong move. But if you just stand in place, then you're not going to know what the next step is going to be. And we have what I like to call analysis paralysis, right? And nobody wants that. Because, you know, if you get in the car and you tell your GPS, I want to go to that place, there are two pieces of information that your GPS needs. One, it can read it, which is where you're at. And the next one is where you want to go. Without knowing where you want to go, then you're just going to get in the car and maybe you'll get there. Maybe you'll get mugged.  So you want to have those pieces of information. Where am I right now? What do I have to offer? One of my favorite quotes says that, do anything you can with what you have from where you are. And so that's what we're going to do.  You've got to get moving, OK?  And I don't know about your GPS, but mine is very annoying. If I don't make a U-turn, I mean, it's like, can't you see, it says no U-turn right there? Well, she can. But, you know, she annoys me to know, and make a U-turn. I told you, make a U-turn. So, yes. So as you move, you'll see what's working. You'll see what's not. You'll see if you need to make a left turn or a right turn. So I hope this analogy is clear for everybody. OK. And my accent. How's my accent? Thank you. So let me tell you about when I started blogging, it was 2005. And I was blogging about marketing and web design and, you know, reaching your clients online and all of that stuff that we all love. And it was fun. And it was exciting. And I was getting, you know, really good traffic. And I was getting clients for my web design business. And it was all really fun because that's how I generated the funds to actually stay home and raise my girls. I am very grateful for that journey. However, in 2009, I had an epiphany. I started kind of adding a little bit of my personal things, my stories to my blog. And I noticed that whenever I share something about being a mom, about my kids, about the things that I was struggling with, or the things that really mattered to me, then my blog just spiked. The traffic, the engagement, everything. I even got opportunities to go on blogging tours and things like that, which are really cool. And you can ask me all about because I love them. But one thing that I did is I aligned my blogging with my passions. Instead of blogging about the things that I knew about and that I was skilled at, I started blogging about the things that truly mattered to me. I started blogging about the things that I couldn't stop talking about if somebody just hit me in the head, OK? The things that I dream about at night, the things that wake me up in the morning. So that is one thing that will guarantee your blogging success. Is that ES is cool to give information, to give inspiration, entertaining. All of those things matter. But when you're blogging about the things that matter to you about the why in your business, in your life, that's when you're going to notice that people are going to resonate with that content. Actually, my daughters, did any of you see my daughters speak? OK, can we clap for those two young ladies to stand up? So those two were the reason why I changed my content around. Yesterday, they were talking about how we need to be authentic with our content, how we need to be vulnerable. So they are my why. Let's see. OK, there we go. So before I talk about this picture, I'm going to tell you that there are two things that are wrong with that picture. The little circle over here is what Simon Sinek. How many of you know Simon Sinek? OK, Google him, or actually YouTube him. He has to talk about the golden circle of the why. Why is the number one question you need to ask about in your business? Why you do what you do? Why are you passionate about it? Why do you need to do it? Why does it need to come from you? Why me, why now? Those are the most important questions in your business. Then the how, and then the what. So it all starts with why.  Well, the first thing that is wrong with that picture is that it is Photoshopped. And it is Photoshopped because this was added on later. And I'll tell you how that came to be about. It was 2 o'clock in the morning in a very cold Florida October night. I had just moved from California with my husband across the country. And Alicia was two, and Alyssa was one. And I woke up with a very weird feeling and I woke up with a very weird feeling in my stomach. And I went and scanned the room. I was living in a room that was about this size. And if you're from Florida, of course, all of you, I think, I live in Texas now. In Texas, nobody knows what an efficiency is, but that's where I was living with my daughters until we found the home of our dreams. Well, I found out at 2 o'clock in the morning that their dad left a note saying, it was too much responsibility.  I decided to pursue other things. So that was the pivotal moment of my life because I always say that there are defining moments in your life, moments in which you either define the moment or let the moment define you forever. And so that's what's wrong with that picture. It was just the three of us for a while. And that is why I started blogging. It was because it was an outlet for me to actually be with my daughters full time, to not have a nine to five job, to not let other people raise my kids, to be able to share with them the things that I have learned. And so I'm really, really happy that I have been able to do that, but I'm really, really humbled because that was instead of, you know, I saw it as a tragedy. And of course I did what every person does. I wanted to cry and I crawled in the fetal position, but I discovered that I had the best part. And I became really grateful about that opportunity. And that's why I'm here. And that is why when I decided to focus on helping other people, I want to help moms that feel scared, that feel that they are worthless, that they are hopeless, that they don't know where they're going to go next or they don't know how to do things. And so that's why we need to focus on the why. If I were to focus on the how I was going to make it as a single mom with no family, I couldn't go back to my country with my kids because it would have been kidnapped. So I would have probably just died because I didn't know how, but I knew why. So ask yourself in your business, in your life, why? Why do you need to blog? Why do you need to create content? Why do you need to take the next step? Why do you need to be successful? And who are the people that you're going to impact with your content? Because in this moment, it was not about me. And my self-worth was probably non-existent. It was about my kids. And then it became about these moms. And one tip that I'm going to tell you is that if you're terrified to create content and to publish, publish is like the enemy, right? One thing that I do is I have one person that I blog for. And she's my imaginary friend and her name is Amelia. Yeah, I'm like a kid. I'm a kid size, so it works for me. Yeah. So I think about her every time I sit down and pour my heart. I don't think that millions of people out there are going to read my blog. I don't think about anything but that person that needs to hear those words. And it's true, the world needs to hear your words. But if one person hear those words and says, you know, thank you, it makes it all worth it. So yes, there's a Photoshopped guy into my picture and a little baby. And so yes, we have a happy ending. So I remarried three years ago and she's two, we call her little pigeon. And she is very active. She's like a little version of me which is a good thing but an exhausting thing. I already have two gray hairs. Which takes me to the second thing that's wrong with that picture. I want my hair back. Pregnancy can do little crazy things to you. So I'm getting there. Okay, so any questions before I tell you what this is? Because I wanna hear from you, okay?  And it's something that I learned from one of my mentors, J. Conrad Levinson. I'm a Gorilla Marketing Certified Master Trainer. And what that means is that J. Conrad Levinson who created the Gorilla Marketing brand and sadly passed away two years ago, he was my mentor and he trained me to do what he does. And basically he used this plan with Steve Jobs with Michael Dell, with people that in the industry and Fortune 500 companies that have very successful solid business, they started with this little Seven Sentence Plan because he believed that your intuition is more important than your knowledge. And actually that Albert Einstein also said that imagination is more important than knowledge.  Because he said that Steve Jobs shared with him about five years ago, because that was three years before he died. He said, I still go back to that sheet whenever I have a question to make a decision. So the first sentence you're gonna write is the specific purpose why I blog is. And if you're not blogging, that's gonna be why I will blog, okay? The specific purpose why I blog is.  And this is best done in five minutes. If you're like me, I want to analyze it and meditate on it and then write three paragraphs and see what happens, but this is something that we're just gonna do on the fly and then you can't fine tune it, but it's better if you just go with it. The second one is the competitive advantage that I'm going to focus on or that I'm going to stress on is. And that is what makes you different. What is the thing that you have that no one else has? And that doesn't need to be a degree or an expertise or anything like that.  Then you're going to write the marketing weapons. Remember it's guerrilla marketing. So the marketing weapons that I'm going to use are. And one thing about this, about the marketing weapons, those are the things that tactics or the tools that you can use to promote your blog. And one thing about this is that we always want to say, you know, well, if I did video, my blog would explode or if I did more keywords and more SEO, my blog would explode. But I want you to focus on something that you're already doing because that is a tool for procrastination. We say, well, when I get a YouTube channel, then I'll start doing it. Or when I hire the guys to do the SEO, then I'll start doing this. But when we focus on what we have right now, then we have no excuses. We could already be doing it. If you have a Facebook, then put Facebook on there. If you have a neighbor, then tell it to my neighbor. Okay, so we all have things right now that we could be utilizing that we don't need to postpone or procrastinate marketing ourselves or creating content. Then the fourth sentence that we're gonna write is my identity. And I know you can't read that, but. Identity means what you stand for. And that was, that's what people call your brand. Yes. Identity is what you stand for. Oh, my identity is. That's it. Yeah. And these slides are available to you too, so that you can get back.  I also, I'm very reachable. So if you wanna reach out to me after this session or whenever, I love to help people blog and take their ideas and share them with the world. Okay, so the six is the main action that I want my reader to take. The main action that I want my reader to take. Yes. Five, right? That's number six. Five. Okay. Five is my identity is. Okay, four was my marketing weapons. That's what I was telling you about. Oh, I skipped number three, okay. I'll leave it for last. Don't worry about it. They're not important. The order is not important. So after my identity, which is the next one? It's the main action that I want my reader to take. I skipped number three, but I'll leave it for last, okay? I'm sorry. I love that. That means you're paying attention. Yeah, go for it. Okay, so the main action, we all want different things from our blogs, from our blog post. Is it sharing? Do you want them to share? Do you want them to comment? Do you want them to subscribe to your blog or download something that you have on there for them to download? Do you want them to hire you? Always know what the main action that you want people to take is and take that across your brand. Have that same call to action everywhere on your marketing.  They don't do what you want them to do. There's too much, too many messages. So you want to focus on one action and you want to always go back to what's that action. The next one is the way you will measure success. And of course, we don't like tracking and measuring and testing, but that is the secret to success, not only online, but with any business, because that's how we learn. It's with feedback. It's with tracking the things that we do, what went wrong, what we could do different, what we need to keep doing, and maybe add something new. So how will we measure success? The one that I left that was number three and there is no particular order, but it was about your audience. Who is my target market? A lot of people tell me, wow, you have 44,000 Twitter followers. How do you do that? Am I supposed to be on Twitter? And I said, well, are your people on Twitter that you would try to reach? That's the first question. If you're supposed to do something, it's because the people that are on that platform want to receive your message. Pinterest, people ask me, am I supposed to be on Pinterest? Well, if you're targeting to women and to moms, then yeah, that would be a good idea. LinkedIn, well, it depends whether you'd be to be or be to see or whatever platform you have. So it's all about the audience. Who are they? And when we focus on our audience, people, the whole model was demographics and that's still important, but we want to focus on psychographics. And what that means is what do they need? What do they want? What are the questions that they're asking? What are the things that they want to know about? What are the ways that they can solve, you can solve their problems? Because really that's what it comes down to. People buy from people they know, they like and they trust, but they do it because they have an idea that buying that product or that service or reading that blog is going to help them either achieve a goal or avoid an outcome, solve a problem or feel a need. Those are the only reasons why they do it. So if you're blogging about yourself and you're not really taking into consideration what your client needs, what your reader wants, then there's going to be a disconnect because they're not going to feel like it's valuable. And with blogging it's all about value. Okay, there are a couple things written on here. That is what I think a content strategy needs to have. There are three pieces to a content strategy. You start with the results first. So what are the results that I want to get out of my content strategy? Then you establish the roadmap, how you're gonna get there. And then you measure your ROI, your return on investment. Yes, that's how you get a strategy together. So the first mistake is not having a strategy.  Because usually we don't start with what we want first. We start with the how. We start with I need a website, I need a Facebook page, I need an Instagram account, I need to have 10 ideas on what I'm gonna blog about, but we don't really harness on the results that we wanna get. I want to reach 10 million moms in the world. That's my results, those are the results that I want this year. And so how am I gonna know if I did that? Well I go back and reverse engineer how I can do that. If I, on December 31st I had reached 10 million moms, what would I have to have done in November? Where would I be? And where would I be in August? And where would I be in June? And what am I supposed to be doing right now? So that's how you get there. What is the roadmap? Okay, so moving along, reason number two is there is no focus on your content. And whenever I work with people and they say, well my blog is not working, the first thing I look at is at their categories. They have like 20 categories that they blog about. And I recommend that you have three categories. Because it's doable. Because it's something that you can set yourself up for success. And you can blog about anything that you're passionate about on those three categories. But if you take all of your categories and you say, well if I were to blog about all of that, where would I put it? Okay, one thing that will help you with is to know that you're really not putting all of the same eggs in only one basket. So I have three categories that I blog about and I blog three times a week. So I know that I'm blogging each day of my schedule. I'm blogging on one category. So my content is consistent and it's predictable.    Some people only go on Mondays. And I know who they are. Because that's the only content that they are interested in on my website. And they subscribe only to Monday.   So it's gonna really solve that puzzle. Oh, what am I supposed to blog about today? I don't know. So it's going to help you organize your content and it's going to help you get that focus. And we already talked about the niche which is the people that you want to serve and how you want to serve them. If you know that you're blogging for moms, like in my case, or you're blogging for photographers, or you're blogging for kids, or you're blogging for WordPress users, you want to speak to them in their language. You wanna know that, yeah, anybody can read my content.  Because it's a blog for moms and it's going to be purple and it's going to talk about kids and have kid analogies and stories. So you wanna make sure that when they get there, they say, this is where I'm supposed to be. This girl gets me. She knows exactly what I'm looking for and she's speaking to me. Speak to one person.  Now say, you know I went on a road trip. It's eye to you when it comes to blogging. Don't write in the third person and write in present tense, present active tense. Make sure that you're taking them on a journey in your story with them and include them. Ask them questions.  Like I said, if you're talking to one person, you're gonna make it engaging because it's a conversation. And I write like I speak. So let me give you this a few things because we started late, so. But the next thing is creating an editorial calendar and take a picture of that because all you need to do for an editorial calendar, that sounds like a big thing, but all you need to do is create an Excel sheet or a Google spreadsheet or office.org spreadsheet, doesn't matter. But just create those columns. And actually there's a blogging also that you can use. I find that I don't ever look at the blogging. I forget about it, but.  It's post schedule and just make sure that you write all of your ideas on there and that if you have three categories, you wanna spread them out. The reason number three is basically that you're not promoting your content. And that could be because you're scared of people and the feedback that they're going to do. But basically a blog is different from a static website because there's engagement possibility. People can comment, people can share, people can really have a conversation with you and that is the most valuable thing about a blog is that you have the ability to create engagement. And content marketing is not content marketing without the marketing part. There are two pieces. Creating content and not marketing it is not content marketing. That's just content creation. And so if you want to monetize your blog, you need to create content so that people know that you have that blog, they need to hear from you. So use a sheet. I created this daily action list for myself but yours can look like anything. Just do a checklist. Whenever you write a blog post, did I share on Facebook? Did I share on LinkedIn? Did I tell people across the street? Use what you have right now and it'll start, it'll pick up momentum. But really the most important thing that you need to do is to block time after you create it to spread the word. So what do you think I do on Tuesday? I spend the entire time that I wasn't blogging, I was sharing my content. Facebook groups people, Facebook groups, LinkedIn groups, they want content and you want to make sure that you're sharing that content there. So those are the three things that you can do to get your blog at a better status. Any questions before we have to, because we are out of time? Any questions about your blog? Yeah. It's gonna be on the WordCamp website and I'm also gonna tweet about it so I'll tweet the slides. Any other questions? It's thepositivemom.com. Thank you. Yes. Well you can create content anywhere, like whenever I get hired as a blogger I actually also get hired as an influencer so we could do an Instagram post and that counts as content, it's visual content. So any of the social media websites you can post content on there. Yes. Okay, one moment please so I can hear. What was that? Yes. Yes, yes. Actually I installed the SumoMe share bar on my blog post so if you go to SumoMe they have a free tool, they also have upgrades but so I installed that on my website and I use my own share bar because when you have a share bar and it already has numbers people are more prone to share. So you wanna be your own advocate and fan so if they say oh one person already shared this they are more than likely to share too. So that's like yes and I just go and share it from my share bar everywhere which is a good reminder too when you see the share bar that kinda triggers you oh I better share my own post. And that's funny because sometimes we ask other people to share but we haven't shared it ourselves. Yeah, I have been published in the Huffington's post but basically as a blogger you have the opportunity to partner with brands. So for example I have partnered with companies like Delta, Dove or something like that. They want you to use their product or their company as part of your story. So one thing that Delta did is they flew me back to my country and the Dominican Republic and they wanted me to do something that I had never done there and to share that with my followers. And I shared my journey, I took pictures, I blogged about it and I told the story of what I was doing and why it was important to me and basically they pay you to do all of that because your voice is important. People want to hear how other people are using products and why and it is a very profitable business. So when you share it? No, I started with my own website. Yeah, yeah, yeah, absolutely. And they'll share it yourself or they found you. They find you. So if you're actually, for example when I partner with Dove Hair, I blog about self esteem and teaching my girls to be confident and they have a campaign that also talks about that and I happen to have curly hair and not straighten it. So basically, as you become the poster person for that particular topic, that's why you need three topics or as little number of topics as you can. As you become known for those topics brands are going to approach you. For example, Steve Harvey contacted me, his team contacted me to go to his conference and be one of the bloggers there simply because I blog about being a successful woman and a successful woman of color. So they approached me because they know that I do those things. So as you get known for what you do, as you put more content out there, you have the opportunity for people to approach you and say, what is your fee? Sometimes they don't even offer a fee. They say, what is your fee? How much do you expect to get paid to do this? And all you need to do is associate your name with the brand name. And with that, I wanna say, don't partner with brands that don't represent your values. So you wanna be clear that the message is the same because you wanna be loyal to your readers that know exactly what kind of content you put out there. So if you don't have a story or your values, you don't resonate with that company. I pass up a lot of opportunities that are very, in the high six figures because I don't really resonate with that endorsement. So as a blogger, not only you have the voice to create content, but you also are a talent for media as a media personality. One last question. Yeah, absolutely. Where's the next? I don't know and I wish him very well. They say that even eagles need a push and the mama eagle really, I don't know how she has the guts, but you know, I think that he was my mama eagle because without him doing that, I wouldn't have discovered the strength that I had. I wouldn't have really strengthened my faith and be able to raise my kids in the way that I did. I think that I would still be barefoot in the kitchen. So yeah, so that was truly a gift and God bless him. And we haven't seen him ever since. So it's a good thing. Yeah. When they call, what is it? What was that? When they call, what is it? When they call. You say it. Oh, what is my thing? It really depends because some people, they want you to write simply a blog post, but in the case of Delta, for example, they treat you like a VIP, they fly you back, they pay for all of your expenses. They also pay you your blogging fee. They also wanted me to share on YouTube, on Vine, on Instagram. And I have a very big following. So the more followers you have and the more views, then you have the ability to negotiate a lot better. But I want you to think about that. Some people think that they wanna wait until they have traffic or followers. Sometimes just because you are the perfect person to share that message, it doesn't matter if you have one page view. They just want for you to be an advocate of their brand. And if you're excited about it, and I speak to the brands all the time, they don't care about your numbers. And actually BuzzFeeds just had an article go to their website or go to Forbes. I think they also used it there. They said that page views and traffic is the old way to measure engagement on a website. Yeah. Do you have one page view? Yeah. Do you find the resources to approach them? Like, how do you approach them? They approach you. They approach you. But I wanna say also that there are blogger networks out there, agencies. So if you have a certain niche, for example, there are blogger networks that have to do with people of color or men, that bloggers, or something like that. Approach them, tell them, you know, I'm a blogger in this niche, and they will partner you with brands. So there are many opportunities. You can also do reviews in which you don't get paid, but you get, you know, your laptop for free or something. So there are many ways that you can start monetizing because let's face it, you know, I wouldn't have been able to stay home with my kids if I had a nine to five job. So you would need to own your worth. You, your words are important, but they are also valuable. They are also worth money. I don't think that there's anybody coming. So I've just continued. I'm just answering questions. If anybody else has questions, yeah. I guess it didn't happen overnight, how long? Well, actually I started blogging in 2005 and it was okay, but it didn't start really blooming until I switched to only mom content, which was in 2009. So, but you know, it has ups and downs. Like when I was pregnant, I didn't blog. And actually I think I published only once in nine months because you know, you got to be kind to yourself too. Yes. Yes, I do. Thank you. I'm your cool positive momo matchi yesterday. Oh yes, I know. I have it back here.