as i work on adding pages to my thesis i thought i'd talk about sds page electrophoresis um so i've talked before about ics page and so in the beginning part of the video i'm going to repost the video from before where i go over some of the theories and then i'm gonna give you a practical look at how we set it up and run it um and that sort of thing i know a lot about what this looks like because i've run since i started counting 738 sds page gels um through my phd process and that's only since i started counting um in order to keep track of them better so i number them and then i like number them in my notes so i can just like reference back and forth between like the pictures and that sort of thing but i know a lot of people don't know what scs page actually looks like um all the and then it's one of the techniques that is most important in biochemistry and that you'll probably do if you take some sort of biochemistry lab so i wanted to give people a sense of what it actually looks like practically one of the first experiments you'll probably do in any biochemistry class is called sds page which is a technique that we use to separate proteins in a small sample based on their size well actually it's their length as we'll talk about later sds page stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis so yeah we call it sds page for short but it's important to know what these various components of the name stand for because that helps you understand how the process works so sodium dodecyl sulfate sds is a detergent which is basically an artificial soap it has a negative charge um and it has like um this long hydrophobic or um water excluded carbon hydrocarbon tail and then this hydrophilic water loving head that has this negative charge um and it hangs out in itself form with a sodium ion um to kind of like counter balance that charge but then when you um dissolve it it dissociates so you get the sodium diesel uh so you get the dodesyl sulfate ion and then the sodium ion so basically once that's in the solution it's a negatively charged thing you don't need to worry about probably acrylamide chain so there's long chains of acrylamide and then they get cross-linked together to form this mesh and based on the amount of cross-linking that you use you can get tighter meshes which are really good for separating smaller things so it can get bigger meshes which are great for separating bigger things then the gel electrophoresis part this is how we actually get the proteins to move through the gel so we establish this electric gradient where you have a positive charge at the bottom and a negative charge at the top and we're going to use this sds to make all the proteins negatively charged so that they'll want to move away from the negative charge that you start them at towards the positive charge so we'll have to go through the gel to do that and based on how long they are they will travel at different speeds because the longer ones are going to get tangled up more and thus travel more slowly you're not going to be able to see them as they go through you're only going to be able to see a tracking dye which travels at the same speed regardless it's not attached to your protein or anything so then afterwards what you're going to do is you're going to stain it your gel with um usually with something like comassie brilliant blue um cbb and there are a bunch of different um formulations of that we'll get into that at the end of the um talk so let's take a closer look so in this gel as we said we have this mesh this polyacrylamide mesh um and we're going to send the proteins traveling through it but you have these if you just take a normal mix of proteins in a solution like maybe you did some sort of um cellular lysate so you broke open cells and you want to see what's inside them or you're purifying a protein so you have a partially pure sample but has most of the protein that you want and then some other contaminating proteins and you want to see how pure it is so you want to see what different proteins are in there um or at least what size of proteins are in there and how many different bands you see and that sort of thing problem is these proteins have shapes that's kind of how they work is that you have these long chains of amino acids but then they fold up um into these structures that are actually functional so the amino acids are like the protein letters and they have this generic backbone part that lets them connect to each other and then they have unique side chains or r groups that stick off and they have different properties and so those properties are going to influence how the protein folds because the amino acids with um some of the prop the side chain some of them are small so they can move anywhere but some of them are big and bulky and they can't twist in the same ways some of them are positively charged they want to hang out with the negatively charged ones some of them are hydrophilic so they're wanting to hang out with the water but the hydrophobic ones the water doesn't want to hang out with them so they have to cluster together in the center of the protein so basically the amino acids are the protein folds in a way that accommodates all of those different um things so you get this beautifully shaped protein that's super functional and then if you were to try to send those traveling through the gel it wouldn't work so well because these proteins the shape would get in the way of them traveling um at a consistent rate that you can compare between the proteins because some of the proteins are going to be like compact and some of them are going to be more loosey-goosey and so they're going to travel differently so what you want to do if you want to separate them by their length is you need to actually unfold the proteins or we call this denaturing so we're going to denature the proteins before we put them in and we'll talk about that in a minute but let's look at what happens when we put them in so the longer the gel is going to act um it's going to act kind of i like to think of it as if you have a sea of basketball hoops like basketball room hoops and you put jump ropes you throw um toss jump ropes down through them the longer ones are gonna get tangled up more so they're going to travel more slowly so what happens is when you stop the moving so we're going to be motivating them through the gel based on the charge so when we turn off the charge they're going to get stuck where they were the bigger ones are going to be on top higher up because they will won't have traveled this far the smaller ones are going to be lower down um but first we have to actually get them to move through the gel and so this is where the charge is going to come in so if you think about it some some amino acids are charged um positively some are charged negatively most are neutral um so if any protein you have it's not going to just want to like swim directly through the gel it'll diffuse around a little so it'll just like randomly move but it's not going to have enough motivation to actually like untangle itself from the gel and move directionally straight from the top where you put it into the bottom so it's not going to be very useful so what you have to do is add charge so this is where the um electrophoresis part comes in um so we use like a power box to set up this charge gradient where we have a negative charge at the um top and the positive charge at the bottom and so if you have a negative charged protein all's great it's going to want to swim through but what if you have a positive charge protein it's not going to want to go through and most proteins are like this see like some parts are charged some parts are negatively charged so you get this overall charge but proteins have all sorts of different overall charges and so if you have the charge interfering with how they move then you're not going to be able to compare what you see kind of like how if you had shape interfering with how they moved you're not going to be able to compare the different bands um to get information about how long the proteins are so we need to kind of uniformly um uniformize everything so how we do this is actually really cool it's with that sds so that detergent molecule is telling you about so it has two cool properties one is that it's negatively charged and the other is that it has this long hydrophobic tail so basically when you unfold a protein it's not going to be very happy with you because it folds it that way for a reason it likes to um especially the hydrophobic parts so the parts that the water doesn't want to hang out with so basically water it forms these really exclusive clicks and so it'll kind of gang up on the protein and cinch around the hydrophobic parts um and so those will get clustered together towards the core of the protein if you unfold the protein then all those hydrophobic parts are going to be exposed to the water and they're not going to like that so they might just like clump up or aggregate and that's not going to be very useful because it'll just get stuck in the top of the like the well of the gel so what you need to do is you need to unfold them and keep them soluble um and so the sps does this because those long hydrophobic parts can kind of like glob onto those hydrophobic parts of the protein but then the sds also has that positive hydrophilic head so it's going to keep the protein soluble well it's unfolded and heat is going to help us um because the protein it has all these layers of structure and so the main the overall structure you see so it you could have um is normally tertiary structure so quaternary structure is if you have a protein with multiple subunits um but all proteins have a tertiary structure which comes from secondary structure which involves the folding of the backbone and then all of that comes from the primary structure which is the amino acid sequence we talked about and so heat is going to help denature that and then the sds is going to code it um yeah and then yes then the sds is going to keep it unfolded and give them that negative charge that they need to send them through the gel okay now that we got that theory underway let's take a look at what it looks like and how we set it up um and some tips and tricks and things to avoid um things to watch out for um how to interpret things that sort of thing okay so in our lab we make our own gels um or and well our tech we have lab technicians who will make them for us too which is really great um so i used to make like all my own but took forever now we have like two lab techs so um they make stocks of gels for us and then keep them like wrapped in um wet paper towels and then coated with saran wrap to keep things tight um so this is an eight percent page so this is a actually like we these are new page gels um this is jealous and we also have like tris tricine um and so it's 1.5 millimeters that's the thickness and then 15 well so there's some benefits to making your own gels and one of them is that you can like choose the thickness um and the number of wells so we have like a whole bunch of comb options so we um so we have like 0.75 thickness and then 1.5 thickness and that's determined by the glass and then you can choose the number of cones so for like setting it up you have this glass so you can see like this one's .75 and this one's 1.5 um and then they have the the short glass piece that goes on front and then you put them in these casters and then pour the gel in between in that slot um and then let it like jellify and polymerize and get all hard and then you can move your protein through them so those are like the caster things up top um and they have this little clamp so i don't know where they are right now oh i have a post i don't have a video on it but i have a post on like how you set these up um okay but we're not gonna talk about that now um so you can also buy like precast ones um so the precast ones usually come in like plastic um plastic like back in front and you actually like crack them open to open them so there's little arrows on the side um so it comes with like a tool is you can just do something like this um just to like crack it open but with these uh you don't want to break the plates um but you do have to like peel them apart carefully when you're taking the gel out um but let's right now we don't want to take the gel out we want to leave it in because we're going to set up our gel um so first we need to prepare our proteins and so i found these samples in my freezer um from a protein purification that i did who knows one because i didn't label these very well because it was just one of those things where it was just like using that it that same day so if it's something that i want to keep for a long time then i'll label it better with like the date and everything um so i've already prepared these samples um so what that entails is i have this i use this like 4x um sds page loading buffer that i make and store it in this nice little rack i've made on my shelf so it's always handy um so it's 4x so normally what i do is i make like 24 microliters so i do like 18 microliters of my protein sample and then like six microliters of this um and then i load like 10 microliters but that's just because that's normally what the like the protein concentration range when i'm doing a purification if my protein's really concentrated then i'll like dilute it first or i'll in or a little less um and so typically i like to load like i think about a microgram of protein if i'm like calculating a lot of times you don't calculate i just kind of know based on past purifications and that sort of thing how much to expect um but if you like overload the gel you're just going to like get a smear or you're going to get like this giant blob and it's going to work warp like everything else in the gel and so there's it's kind of tricky when you're doing it based on like um like migs of protein or in this case we're talking micrograms so that's like a thousandth of a milligram um or a millionth of a gram um but so it's like is it if you're just like overall protein then it's like can be a ton of different proteins if it's like a beginning step of a purification say um you have like a ton of proteins if it's like the end then you have like a single protein and so you if you have a ton of proteins you can take a higher amount load a higher amount of protein because all of the individual bands will be less um but if it's like a single protein then you have like a ton of it then it's just going to like warp your whole gel and then you're just going to get this brush thing um but of course if you load too little then you're not going to be able to see like all the contaminating bands and that sort of thing um another thing to keep in mind is that for like a western blot you don't need to load as much because the antibodies are going to amplify the signal um with this though we're just like stain we're just staying the gel with um like a camassi based stain um so it's just like an all protein stain um it's going to stain the proteins and so if there's not very much you're not going to see things um and so you might get the you might not see a protein or that you are looking for or you might um like or that you abandon what you think is the protein you should be looking for or you um you like think your protein's a lot purer than it actually is because you only see a little faint pan for your protein you're like yeah it's there but then there's a bunch of other proteins that this dye is just not sensitive enough to pick up um and so when we load the samples we have these nifty nifty gel loading tips i was so excited when i found discovered these well i mean like i didn't really discover them like i just when we had these in the lab like i didn't have these in undergrad but they have these long tips and so they actually like reach down into the wells and so you can pipe it into the bottom of the well and not have it just like shoe back up or like not try to do it from the top and then have it um problem it's a lot even more useful for um like when you're running urea page gels so like for rna separation because there you don't have like the stacking gel that you have with sds page so that stacking drill that we talked about it has like the bigger holes and so it like everything kind of gets to the start start line at the same time which is where the like the resolving gel starts um you don't have that with a urea page so like where wherever you pipette it in is where it's gonna start and so if you don't get all the way to the bottom of the well then it's gonna mess up but also don't puncture the bottom of the well um okay so we have the so that 4x buffer so we i just like make a ton like a bunch of it and then like i'll equate it so make like one middle portions and different tubes and then keep it in the freezer um but it basically has like sds so it has the detergent it has like a reducing agent it has the dye um if you are really worried about like reducing agents so the reducing agent in here is going to so that's what um keeps things um in a reduced environment so that like the cysteine cysteines don't form like crosslinks and so if you had like a cross-linked dimer it would like um break it up um and so if you like the reducing um agent it will like go bad over time it will just like become not effective so if you're worried about that sort of thing you'll want to add reducing agent brush before you run so you can add like bme um so yeah but the main thing is that it has like that dye in it and the sds so the detergent um which is going to keep the proteins unfolded and slippery but in order to get the detergent to coat the proteins we need to also give them some help from heat so after i mix the dye and the protein give them a quick spin in the little like pull centrifuge and i'm gonna stick them on this heat bath um so we have this like sand bath um so i think it's at like 95 degrees um normally just give it a couple minutes and it should be good and so this is going to help unfold the proteins and let the detergent coat them all a thing to keep in mind is that if you have potassium chloride in your buffer it is probably going to form this like precipitate so you want to like pipet it fresh like as soon as it's out it's going to be like most soluble when it's hottest so you want to like just take it straight out of the bath and load it um so sodium potassium um sodium dodecyl sulfate so sds um so that's soluble but potassium to decimal sulfate so the potassium if potassium teams up with that detergent that's not soluble so that's gonna like get make it precipitate and so you might be like trying to pipette it in and you see all this like clumpy stuff um so don't freak out that's probably the um the kcl in your buffer and so that's a problem with some of my buffers that i've had to deal with um and then the other thing we're gonna want is a ladder so a ladder um it's just like a molecular weight standards so we use this like bio-rad precision prop plus and so this is like yeah so you have this ladder that has like all these like proteins of known size that you can compare to um and so this is an unstained ladder so it's not going to um show up until we um until we stay in the gel so like the dye in here and the dye in the loading buffer those are both um those are both just like tracking dyes so they're not actually binding to the protein they're just like traveling along like the front of the um the liquid flowing through the gel so it's kind of just like showing you the where the liquid is so it's like the the thing that goes in front of the racers whatever like that would be um so you're gonna have to actually stay in the gel in order to see the ladder and see your other proteins but that's gonna um having the ladder there is really important in order to show you like this um general size range where your protein fits in um like it should be around where it should be like in relation to the lines but don't worry if it's not exactly because different proteins will travel differently um especially because we're talking about like length instead of like lobby size and that sort of thing um so some proteins will travel a little differently than you might expect it can also be altered by things like phosphorylation or glycosylation so modifications of like phosphate groups or sugar chains and that sort of thing can uh slow it down um there's various weird things and so sometimes proteins just run a little funny and we never really know why but anyway okay um so what we're gonna do is this is really hard to do things one-handed i'm gonna like try to set this seti down okay so we take the gel and so it's really important that if you make your own gel so you keep them like wrap keep them moist so when you buy them pre-made they normally come in like a little packet with some liquid um and so with us we just like put wrap them in wet paper towels um and so you can see you have those gel plates where the gel was poured in and polymerized then you have this so this is a 15 well gel you can see that that line there so that's where the so you have a stacking gel on top which has those big holes to get everything let everything like travel at the same speed until it gets to this resolving gel so that down here you have um a tighter mesh for the proteins to actually get separated um and so resolve so you separate them so that you can resolve the individual ones so tell them apart basically so you can pull the comb out like vertically as you can some people think it helps to like do it well it's in the liquid but i have better coordination when it's out of the liquid um so now you can see there's like wells where i'm gonna put the protein in um so now i'm gonna stick it in this running gasket thing whatever you call this thing um so these have this is a bio red like tetra cell thing so this is like our buffer chamber or this is going to go into this has like the electrodes um so that's where like the energy like the electricity is going to go and it's going to like the electrons come in and then it's going to like split water and stuff and so um that's that's where the bubbles that we're going to see come from it's like this electrolysis reaction um so you have the splitting of water and it generates hydrogen and oxygen gases um and so you see bubbles um and so the bubbles is a sign that it's working so you always want to look for the bubbles and then get really happy when the bubbles form okay so this have these like you can tap it so that you have two gels running um and so if you don't if you only have a single gel then you can put in like a buffer dam um and so this buffer dam's like a fake gel um and so it says like there was one side that has like this thing so if you look here this like rubber gasket it has this notch and these notches are really important because whatever you put whether it's the gel or the um dam you want to stick it so that it like is right up against that or it's gonna leak so normally i stick it in like the bottom first and then i look and i push it right up to the edge and make sure it's not like overlapping the edge um and it's just like right up to the edge so right tight with it and then i hold that on while i latch this except i'm gonna first i need to add my gel to the other side so but i'm gonna do the same thing where i'm going to put it down to the bottom and then flat and then push it up to the edge and then clamp it so that's really important with these because they have a tendency to leak if you don't like setting it up right um you'll see that the buffer level so we're gonna fill the buffer like up all the way to the top but you'll see the buffer level if it starts to go down then it can't um like do that water splitting very well and the it'll slow down or it might even give you an error message so keep an eye for you don't want it to leak if it does leak you can actually like add more buffer during the run but you don't have to deal with that um so now the important thing is to put it in the box right so thankfully they have it colored nicely um but that is not foolproof um even for like people with phds um i've seen in the lab or people um like me who are trying to get a phd but so yeah so you want to make sure that red goes to red and black goes to black so there's like two slots in the front in the back um so there's some units so this sorry all right so this lid the lids we use most of the time they're actually have they're set up so that they just have for two but um there's oh i guess no these don't work with both but they have we have different so this one so you can see this is if you want to run like four these don't have the sticky things on top and see that's where you want to do it for but if you only have two you want to make sure that you have the sticky things that are going to go in the holes and so right now so you need to put it in the slot in the box that'll let you put the cap on the right way so you have red to red black to black okay so let's set up our gel now so we make our own buffer too so we have like two buffers for this system so we have like a high molecular weight buffer and a low molecular weight buffer um and we also have like a different buffer for for the glycine gels but so i normally start by filling the buffer dam so that part that we just made and to make sure i do this first to make sure that it doesn't leak and then if it looks okay so now i just keep pouring and so you want to make sure that you cover the bottom but you don't need to like fill it all the way up or that sort of thing um so now that we have the gel set up we can actually load it um and so the bad thing about those gel loading tips is that they only work with certain pipettes so this one works um okay i don't know if i can do this sort of thing one-handed can't even open the gel box the things i do first i come right okay so let's start with the ladder so i'm just going to like load 10 microliters i don't know if i can do this very well on camera i'm nervous but so it can be kind of hard to see the well sometimes but you kind of get um good at it over time and some of the pre-made foodcast ones actually have like guys on them um like limes on them my biggest problem right now is just doing it from the back okay so if all goes well if i don't know if these samples are still good or if they got degraded but they're from a purification so we should be able to see the samples like things get less fewer bands over time because each protein will have its own band and then that'll show up yeah it's a lot easier to load from the front than from this awkward angle but you get the point okay so now i am going to stick on the lid and this is not the lid that was attached to the box this one was attached to the box so now i've set this up and i'm going to come and i'm going to turn on this power box i'm going to start out at like 150 volts so the voltage and stuff is gonna depend on the gel uh type you're using and that sort of thing so look to a guide and don't just go with what i say um we'll start with 150 okay i'm going to press start but i want you to look at the box not at the start thing because something cool is going to happen well it should okay let's press run bubbles you see the bubbles i don't know if i have the camera right yay okay bubbles so bubbles that remember that bubbles are telling us that that electrolysis is working and so you're having that water splitting which means that there's like electrons flowing through things which is going to create that charge gradient um so that are now negatively charged proteins thanks to the sds are going to travel through to the bottom which is now positively charged thanks to that electrolysis stuff um and so you can see that the um where we put in our samples so the dye is kind of starting to migrate um to that stacking line or the resolving gel line um and so it's all going to get kind of clumped up there and then it's going to travel start traveling through um and so now it's going to run for like 45 minutes or so i'm not just going to like sit here and watch it and i'm not going to make you sit here and watch it um so i will stop the video and restart it once the gel is done okay our gel should be done now um so the you can see the dye line is almost to the bottom normally i let it go all the way out but people are gonna be actually coming into the lab soon um and so i'm going to stop it now um so i just i'm going to turn off the power box and now what i'm going to do is i'm going to take the gel out and actually what i need to do is get a gel staining box okay so i'm going to get a gel staining box so like some of these are just like sandwich containers some are the tops and bottoms of old tip boxes um my favorite ones are like these um like square ones that are actually i think they're made for this so they probably charge you way more um okay so now i'm going to take the gel out and i don't know how best to do this and tape this try this so what i'm actually going to do is first i'm going to get some sticks this is like an instant blue stain so we've been trying out a bunch of different ones so the one we used to get got discontinued but it works so much better but anyway it's like an instant stain it's based on komasi which is like the classical so the classic like massey is like this super dark thing and it um stains really nicely but it also you have to like destain and you have to fix it and stuff it so that's so the instant stamps are really nice because you can see things it has less like background and that sort of thing and you can like reuse it a bunch of times so like in the fridge [Music] i'll actually since this is just a demo i'll just use one of these on past ones because i don't really care okay so yeah you can just like after you use it you just like pour it back in here and reuse them like tons and tons of times um but so sometimes it's nice to actually pour your stain first because it um like then put your gel into liquid and it will hopefully um prevent ripping better um so i'm just going to like pour off the buffer i can just open it up and so now because this is one of the glass plate ones so i don't need to like crack it open i just want to gently peel it open so like find an open spot and then like gently peel it open it'll want to go to like one side or the other and just like let it um sometimes it'll flow out easier just like find a bottom corner gently peel it off you can see hopefully not rip it by trying to show you but yeah it's just this really thin thing that's peeling off so now it's yeah um and so you can see that there's well you probably can't see based on the view but there's like a line you can see the stain from the like that's just like the running dye because it didn't go all the way off um but the proteins actually up above it but we can't see it yet because it might be called an instant stain but it actually takes a couple minutes at least so well that goes then what i do is i like clean up um so since these plates are reusable i just wash them off with water actually we reuse the we reuse basically a lot of the stuff we use to run sds paid shelves which is good because if like i alone have run like 738 or 739 now um so yeah so we reuse so we make our own like writing buffering we also re um use this we just like pour it back in the bottle um so yeah so we're really lucky that we have great lab techs um and so they will like make a ton of it so we actually keep like um five times uh running buffer in this big thing uh okay let me just like change my glass really quick [Music] okay i'm back and let's slap me um so i can take my phone and actually show you so we keep this like five times running buffer um and so then this 5x so like 200 mils of that and then fill up the bottle to a thousand mils at first um i was like super making it super exact and stuff but it's actually doesn't matter too much so um but yeah um so i can just like pour it back into the bottle so we have like a funnel that'll help me um so now the protein so it should be beginning to stain but it's not so protein should like just begin appearing it's to make it easier to see we have like this light box i don't know how it's going to show up on the camera but so we can put our protein um onto the light box um so it it's easier to see things if you actually put it directly on the light box but then there's more risk of ripping it so i can see the ladder but i can't and i can start to see my protein bands but it's going to need a minute so i'm going to clean that up and then i'll come back to you guys okay so i got pretty sidetracked uh with some papers and then like tracking down the author of the paper and like emailing this person in germany so fingers crossed they get back to me uh because they have a cool um there's some cool methods i was interested in which is just uh i'm always saying this because it's just because people don't often know that you could like students don't often know like you can actually like email the paper authors and um hopefully they'll get back to you and answer questions and stuff so i've emailed a few um before and it's worked out really well um so don't be scared so for the staining uh well staining we keep it on this like rocker shaker platform that creaks really loudly especially if you have too much stuff on it so sometimes we have like a ton of boxes on there okay so now let's turn on the light okay well it got flipped over so you can see like if it was up the point seven five wanted to be really living okay so one of them clearly had like way less protein in it because i can't even see the band um but so the first this lane is like pre purification and here's at the end and so you can see that this one has like a bunch of little proteins um and so it's less pure so you can see like in this one you still have a little bit there look at this little contaminating protein but compared to the main protein it's a lot less so in this case you have the main protein then you have all these other bands and the proportion of the main protein is less um so if you look at the ladder so you can see that when we looked at the gel my the protein was like between the 75 and 100 which is perfect because my protein is like 97. um and so yeah so the more so each band is going to represent like a single protein or like multiple proteins that are like the similar length um and so this the stronger the band like the more there is um but like it's deceptive because for like small proteins you can like the same amount like the same number of copies of that protein so like the same um molarity or whatever it's gonna have like a weaker signal because the how the dye works so it's like this non-specific dye that just like kind of globs on to some of the protein um lettuce some of the amino acids and so it can also differ depending on how many like the cup the amino acid composition of the protein um so it does more or less with the ones that the comassie likes um and so yeah so but the stronger the band in general the more of that protein there is and then the more bands there are the less pure it is um so when you're doing a protein purification you want to like end up with as few bands as struck possible and they should be strong but of course how strong they are also depend on how much you loaded um but yeah so right now the gel like the background if it is still kind of like bluey so if i wanted to make the bands like crisper and clearer i could do is i pour off the stain and then i put it in water and then put it back on the shaker and then the like the background will get clear um and then it's easier to visualize things and then we could also we have like a gel image or thing that we can take pictures um so we don't have to keep the gels forever although if you keep the gels forever it's kind of cool because they get all shrinky dinky i don't know if you guys like shrinky dinks but yeah so this makes me really happy so two happy things bubbles and shrinky dink gels so hope that helps you um and have fun running your pages um so they're a lot more fun probably on their first ones than in the 739th but well actually in the first ones are probably just too scary um but don't be scared um just make sure you put red to red and black to black or under red um and yeah so have fun