welcome back to our tour of protein purification techniques next we're going to look at ion exchange chromatography this one is very applicable and widely used and this is separation based on charge differences so the idea with where our charge differences come from in a protein is the same as what originates from the amino acids just like amino acids have an isoelectric point so it is our protein so the pi or the isoelectric point for our protein is the ph at which our protein is going to have a net neutral charge okay so what does that mean what means the charge on our protein is zero how can we have a protein with a charge of zero well it means that our deprotonated carboxylate groups are going to equal the protonated amino groups and because they're equal this is going to give rise if we add them together or some of them gives rise to a zero charge the isoelectric point for a protein is the point at which that protein is going to be the least soluble because these charges can find each other the protein can aggregate together and instead of interacting with solvent when it has a net charge on it it's going to aggregate together these charges will find each other and it clumps up and it falls out of solution whereas if you have an overall charge on your protein it's going to be very soluble because of those net overall charges those net charges are going to repel each other and push the other like charged molecules away and they will have a nice solvation sphere of water around them at a ph less than the pi so we're gonna have two different i'm gonna separate this into two pieces on the left we're gonna do ph less than pi and we can think about this then in terms of increasing ph and then on the right side above our pis the ph that is greater than or above the pi okay so we know when we change the ph for our amino acids that we change their charge and because proteins are made of amino acids as we change the ph we're going to be changing the net charge that's on the protein at a ph less than the pi we're going to have a net positive charge on the protein okay why well because at a ph less than the pi we'll have more protonated carboxylate groups so our carboxylic acids are going to be more protonated and they have a net neutral charge and then our amino groups will be protonated and have a plus one charge so the net positive charge is coming from the protonated states having a plus one and our carboxylic acids being protonated and being neutral as we increase the ph so now we can think about this as increasing ph as we go across the bottom here as we increase the ph our carboxylic acids will be deprotonated first because again this proton has a p pka that's going to be much lower than that of our amino groups in addition we have histidine that's in the physiological ph range that will also become deprotonated and we'll start to lose some of our nh3 plus charges and become nh2 above the pa our carboxylic acids will be deprotonated so they have a minus charge and then our amino groups will start to be deprotonated as well so instead of having nh3 with a plus one charge our amino groups will have an nh2 charge thus an overall net negative charge above the pi and so understanding this we can start to understand why and how we can use these separation techniques for differences in the charges of our protein there are two types of ion exchange chromatography because we have two ions right so we have a cation exchange chromatography and we have anion exchange chromatography okay so when i say cation exchange i'm talking about the resin that's in my column is going to be made to bind cation so a cation exchange column binds positively charged ions so we name it for what binds the resin is going to be the opposite charge so the resin is going to have a negative charge and it's going to allow for the opposite charge to bind then to the resin an anion exchange works the same way but opposite so an anion exchange resin has a positive resin positive charge and it's going to bind negatively charged ions to it so the negatively charged ions bind and the resin is the opposite charge so the resin here for anion exchanger has a positive charge the two most common resins that are available for ion exchange chromatography are d dea cellulose it has a possibly charged resin therefore it's going to bind anions and this would be an anion exchange resin in a similar fashion the cm cellulose is a negatively charged resin so it's going to bind cations and this will be known as a cation exchange resin there are other resins available these two are just very common for doing iron exchange chromatography if you go out to biorad's website so bio-rad is a very common company that provides research materials for the life sciences and you'll see that they have some other types of resins and when you do your protein purification techniques online you're going to see some other resins that are also available i'm going to have you look them up so you get a little feel for the type of resins that these are okay so let's talk about the way that this chromatography works and how we can apply it this is very common technique great one to know the first thing that you're going to do when you do iron exchange chromatography is first you're going to pour your column or your column if you order it might come pre-poured so pour column with your resin if you're working at a big company that does this technique over and over and over or a research lab that uses this technique common commonly you might be working on an instrument that is automated and a column that is already pre-packed or you pack it yourself um and then proceed with a whole setup that does everything for you at an undergraduate lab of course we don't have the money for those because those instruments are tens to hundreds of thousands of dollars when i did this in grad school just our service contract was 25 000 and that was many years ago we won't put a date on it but just know that these are very very expensive instruments and the upkeep of them is also quite expensive so when you think that oh north park is poor because we don't have one well no undergraduate graduate lab is going to have them because it's simply not feasible financially for the amount of time that we would use it we don't get that money back out at the same time these resins themselves are very expensive just a little bit of resin when i order one of these from bio-rad you know and maybe go look at the prices when you look up these techniques because you might be surprised at how expensive they are okay so first thing you're going to do is pour your column with your bezel you usually will put a little piece of like either a filter at the bottom or a little piece of cotton that keeps the resin inside your column so the resin is not going to pour out the bottom and then what you're going to do is you're going to wash this with buffer and there might be some other protocols for washing but you definitely want to wash this with the buffer that your protein is in and then you're going to load your protein mixture so we know that your protein is contaminated with other proteins and we want to separate your protein of interest away from the mixture so we'd load the protein mixture on the column and then we're going to wash our column okay so a protein is loaded awesome now we would wash the column okay so let's talk about what happens when you load the column okay so if you look now at this figure that i'm showing on the left this is a cation exchange column how do i know that well because the the resin is negatively charged and it's binding these yellow positively charged proteins to it so this is named for the ion it binds so this is a cation exchange column maybe it says that somewhere probably don't know okay and so what's going to happen is the ions that are the same charge as the resin are not going they're going to repel each other they're not attracted to each other and the same charge of protein with the same charge as our resin they are going to flow through and then our cations any of our proteins that are positively charged are going to bind to the resin and be retained on the column okay so these red i don't know balls globular proteins that are negatively charged these are going to be found in your flow through and you always want to keep your flow through so the flow through are the things that don't bind then you're going to start washing your columns so you'll do first a buffer wash the buffer that your protein is in to wash your columns so that all these negatively charged proteins or if there's other you know small molecules that we don't care about that are in there we want them to come out next we have two ways to elute the protein so we're going to elute the protein which means release the protein from the resin and the way that we're going to elute our protein is one of two ways one is more common than the other we could change the ph and use a gradient of different phs to elute the protein or we could use a salt gradient if you're in class we might talk about why one might be better than the other i've only used one of these techniques to allude protein and that's the salt gradient so i'm going to use an increasing concentration of salts to elute the protein okay why would i do that well keep in mind if you let's go back up to this idea of our pi and our net positive charge below the pi our net negative charge above the pi and remember that as you change the protein ph you are going to change the charges on the protein which means if you're at a pi or your ph is less than the pi and then you increase your ph such that it becomes negatively charged and it would be released then from a cation exchange column because remember the cation exchange column is binding it at a ph less than the pi okay so this is when a cation this would be its cationic state and this is when it would bind to a cation exchange column when it's at a ph less than the pi okay but if we do that and we go through the pi the pi is the least soluble your protein is going to be so if you cross through that pi there's a good chance that you could aggregate your protein and precipitate it out on the column which means your protein is now stuck on the column and you're not going to get it off not not very easily anyway you're most likely going to have to use a sodium hydroxide or hydrochloric acid wash to try to get that protein off and even then it can be a real pain and you can actually clog your column so i've never used a ph gradient and that's why instead we're going to use the salt concentration okay so before we do that let's cruise over here and denote that at a net negative charge when you're ph above the pi this is when you would want to use anion exchange chromatography or this is when it's going to bind to an anion exchange resin because it has a net negative charge okay cool next so we're down here step five we're alluding our protein with a salt gradient so you want to start with a low gradient and increase it to a higher gradient and if you do this slowly what you're going to see happen is these proteins all of the cationic proteins are going to bind to the column but those with that are weakly cationic are going to be released at low salt concentration and those that are stronger cations will be released at a higher salt concentration meaning that you're going to have an usually a fairly nice separation using a difference in ionic strengths to release these cations from the column so all of the cation that binds hopefully yours will separate out nicely or the protein that we're interested in will separate nicely from the mixture so we usually start with like a zero percent salt concentration and go up to like 0.5 molar an acl is very common but if you have something that is very ionic has lots of charges you might go all the way up to one molar and acl so it really depends on the protein and what works best usually once you find a way that works you don't reinvent the wheel so to speak you stick with it because why waste time trying to perfect something when good enough gets the job done and usually at the end of the day you might couple both of these techniques together or you're going to couple it with another technique anyway rarely do you just do an anion or cation exchange column and be done with your purification you're usually going to do something after that okay let's talk about what happens after we allude our protein so after we elude our protein or when we elute the protein we're going to collect fractions okay so we're going to collect fractions so we have a variety of tubes that are set up and no i'm not a great artist and these are looking like little infants wrapped in you know the hospital i don't know these are tubes totally tubes and here's our fractions one two three four five and they'll go on right and they again if you have this animated you have a fraction collector that's going to do all of this for you all you have to do is make sure that it's working because if something's going to break it's going to be your fraction collector when these proteins are eluded oftentimes we're using some sort of detector again if it's automated which very frequently is uv vis proteins are going to have an absorbance at 280 nanometers so when you reset your results for the dna lab and in that the directions were to look at the protein concentration at 280 nanometers and then nucleic acids at 260 nanometers because the proteins that have aromatic side chains will give a nice absorbance at 280 nanometers okay sometimes your protein is colored and it's fun because it's pretty and you can watch it dilute from the column and that's extremely helpful but that's not always the case there's other detection methods too but this is going to be the most common so once you elute your protein you can if you don't have a detector set up you can take your uv at 280 nanometers you're going to find which fractions have proteins in them and i'm just showing an example of five fractions you could have 30 or 35 or 150 fractions it just depends on how long you run your column and how big that column is so it has more to do with the size of scale up than anything else okay so you'll detect which fractions have protein and then you want to know which fractions have enzymes fractions have protein in them and then which fractions have the enzyme of interest or protein of interest all right so the next thing to do if you don't have a clear way to see it visually i like i'm a bioinorganic chemist so i work on proteins that are generally colored and it's very easy to visually see which fractions i'm definitely keeping and then i might do a 280 and look at perhaps another peak because most of them have a metal that also has another wavelength that they absorb at and then i can decide on that based on the ratio what ones i want to keep so work on a colored protein right it makes things easy but if you don't know then you need to do an assay and you're going to do a check whatever you're using to specifically identify your protein you would assay and keep those fractions so after that then you pull these fractions because they're going to likely have a high salt concentration from the ion exchange chromatography then you would couple this with dialysis or you might do a gel filtration column which is going to get rid of your salts that's the idea here is just to do a clean up method get rid of the salt and then you might couple this to another technique if it's not pure enough after this to check purity gel electrophoresis is a great technique okay that's a lot of information these techniques are very widely used and some of the most like difficult to understand from students perspective so i wanted to give a very clear even though this is longer it's not quite our quick short intro to technique but it definitely gives you more information here and especially understanding this up here of where the charges come from okay so keep in mind amino acids change to their charge with the ph change and therefore we can use this to our advantage and separate out based on differences in charge [Music] you