Tiny columns can purify tiny amounts of protein, but I doubt tiny AKTAs can – but they are oh so cute! (and the best marketing freebee I’ve ever seen). So GE, if your goal was to get me to do a post on ANALYTICAL SIZE EXCLUSION CHROMATOGRAPHY where we use tiny little columns to separate tiny amounts of protein and see if they interact – mission accomplished….
Proteins are molecules that serve as molecular workers and they’re made up of long chains of amino acid “letters.” These letters are like “building blocks” that have generic backbones that let them link together through peptide bonds and, because the different amino acids have different unique side chains with different properties, they cause the protein to fold up into shapes perfectly suited for carrying out various tasks.
There’s a huge cellular advantage to making proteins out of premade parts – it’s quicker and easier than having to make everything “from scratch” each time. Imagine if a car factory waited until it got an order before even finding the nuts & bolts & cutting the metal.
But from a protein biochemist’s standpoint, the similarity makes it harder to separate them and see what’s what because different makes and models” have the same parts so – Kinda like how a recall of one car part can affect multiple makes and models but a recall of a boat part won’t affect your car.
For example, unlike how you can add DNase (DNA chewers) to remove DNA from protein preps or add protease (protease chewers) to remove protein from DNA preps, if you add protease to protein preps, you’ll chew up the protein you want along with the one you don’t.
So we need different ways to tell proteins apart. We commonly use a type of technique called PROTEIN CHROMATOGRAPHY that separates proteins based on how different proteins interact differently with different types of resin (little beads) that we fill cylindrical glass or plastic columns with.
There are different types of chromatography and a lot of them involve the protein you want sticking to the column under initial conditions (due to charge-opposite charge interactions in ion exchange chromatography or some specific feature like a genetically-engineered affinity tag in affinity chromatography). If your protein sticks but others don’t, you can wash all those others off and then change the conditions so your protein gets pushed off.
To help make the process easier, we often use a liquid handling machine, like an AKTA. I’m in a protein biochemistry/structural biology lab so we do a LOT of protein purification, so these machines are in high demand. Thankfully, our lab’s super fortunate in that we have 3 of these – and are getting a 4th in a couple weeks!!!! These machines are FPLC machines. FPLC stands for fast protein liquid chromatography & it uses pumps to push your proteins through the column. More here: http://bit.ly/30LklxG
Proteins need a “push” to get through the column. When we use gravity flow in hand-packed columns, we rely on gravity alone (which can take a really long time and often has to be done in the really cold cold room). The FPLC gives gravity a hand with pumps that provide some push (at a flow rate you can control way easier than fiddling with stopcock angles).
By controlling the pump settings you can control how fast the liquid flows BUT NOT (at least not directly) how quickly your proteins move through the column since each protein will interact with the resin differently, which is the point – if they didn’t you wouldn’t be separating anything just moving the proteins from one place to another.
Speaking of interacting with the resin, unlike with affinity chromatography where the proteins actually bind (reversibly) to the resin, with size exclusion chromatography (SEC, aka gel filtration (GF)) the proteins don’t actually directly interact, they just take different routes.
Smaller proteins have to go the long way, so it takes them longer to get through and they elute (come off the column) later – which we see as peaks on the chromatograph that measures light absorption (protein absorbance is typically measured at 280nm.
Imagine you have a bunch of cars you want to separate. Affinity chromatography is kinda like having snow-covered roads. Cars with snow tires and/or chains can go through but other cars get stuck. Then you can add “snow plows” to clear the road so the proteins can continue on their way. The stronger the protein interacts with the column the more “snowed in” it is and the more plowing’s required.
The “snow plows” we add are typically competitor molecules that compete for binding sites (so it’s kinda like the snow plow actually takes the protein’s place – so not a perfect analogy but…). So you can gradually change the conditions to separate the proteins even further.
In SEC, on the other hand, none of the roads are snow-covered, but differently-sized cars have to take different routes. You have lots of cars traveling to the same destination – the fraction collector (who knew it was such a destination location!)
The routes have tunnels with different clearance heights. Bigger “cars” are too big to fit through the tunnels, so they’re excluded from a lot of the roads. So they miss out seeing a lot of the inside of boring beads and therefore they have shorter trips and get to the final destination more quickly.
Instead of concrete tunnels under overpasses or through mountains, the tunnels in SEC are tunnels through porous beads. The beads are often made up of agarose or some other sugar or some polyacrylamide if you need something stronger and/or finer. What these materials have in common is that, from the protein’s perspective, they’re pretty “boring” as is – like well-paved roads they won’t slow the proteins down.
The “rules of the road” in SEC are that cars have to travel the longest route they can – if they can fit through a tunnel they have to go that way – at least those roads are less crowded right?
But imagine you have car-carrier truck. If it’s empty, it’s not that tall, so it can fit through the tunnels. And if the cars it will carry are on their own, they can fit through no problem as well. But put the cars on the truck and now you have a bigger, taller complex that can’t fit through anymore. So it’s route’s restricted. It can’t go through as many tunnels, so it takes a shorter route and arrives more quickly at the destination (fraction collector). This is the basic of ANALYTICAL SIZE EXCLUSION CHROMATOGRAPHY
Usually, I’m using PREPARATIVE SEC – I separate all of my almost-pure protein by size to make it almost perfectly pure and keep all of that precious pure protein. But in analytical SEC, it’s more like PAGE in that you’re using it on just a small bit of sample to “get a look.” SDS-PAGE is denaturing – the SDS & heat unfold (denature) the proteins – so if you see multiple bands you can’t actually tell if they represent proteins that were actually interacting, or they were just both present in the sample. more on SDS-PAGE: http://bit.ly/2EphbGt
But with SEC, you don’t unfold the proteins, so interactions can hold. the interactions have to be strong enough to withstand the journey (the cars on the truck bed have to be at least somewhat held down so they don’t fall off – thankfully in the tunnels there’s not far to go so the cars, if they temporally slip, can get back on fairly easily.
Of course, if 2 proteins elute together it could just mean that they’re similarly-sized. But if they are significantly differently-sized they shouldn’t come out together unless they interact. What you usually do is run a sample of each protein on its own. The smaller protein will elute at a larger volume (later timepoint) and the bigger at a smaller volume (shorter timepoint). When you mix them, the peaks will shift.
A leftward shift in the chromatograph indicates binding – but not “too big” of a shift – if the elution is really early, instead of a nice interaction it indicates aggregation – proteins clumping together so that they can’t go through any tunnels. The void volume depends on the column size
Sometimes, depending on the size of the proteins and the resolution of the resin, it can be hard to see a dramatic shift in the bigger protein’s peak. But what’s more obvious should be a disappearance of the smaller peak. The peak-shifting’s indicative of binding, and then you can take the corresponding collected fractions & run an SDS page to see if you can see multiple bands.
I was just standing in the hall yesterday and our AKTA specialist person came up to me (he was there for something else) and told me he followed me on Twitter and gave me this AKTA LEGO kit and it was almost as exciting as the protein I got (he wasn’t offering that though…). My labmates were super jealous but my Twittering apparently paid off – I don’t do endorsements but I guess like half of my posts are kinda about and/or in front of AKTAs so…