I spent so much time last week getting proteins clean working at this “protein laundromat” – and telling you about the protein chromatography this protein chromatography it was helping me with – that I didn’t properly introduce you – so, what do you see with FPLC – what’s actually going on in AKTA? 

If you’ve seen my lab bench in the middle of a protein purification, you know I can make quite a mess, but when it comes to the protein itself, I’m a stickler for cleanliness. And my main go-to protein cleaning tool is PROTEIN CHROMATOGRAPHY, a way to separate proteins based on differences in their properties. That bench mess can wait while I bring my protein to our protein “laundromat” – a FPLC (Fast Protein Liquid Chromatography) machine called an AKTA. So what’s actually going on in an AKTA?

First off – something that’s always confused me – why’s it called chromatography? When I hear “chromatography” my mind jumps to “colors” – that’s what “chrom-“ means, right? And this always made sense to me because I would think back to those early paper chromatography experiments I did as a kid where you separate the different colored inks in a marker using paper chromatography. But I’m not separating proteins by their colors, so why’s it called chromatography?

The term “chromatography” did in fact come from its original uses separating colored compounds. In particular, Russian botanist Mikhail Tswett in 1903 used it to separate colored plant pigments. Etymology-wise, chromatography means “writing in color” which I think is so poetically beautiful!

But the important part – that which makes chromatography chromatography – is the separation part, not the colors part. You have 2 phases – in protein chromatography you have a solid phase which is the resin (little beads) in the column (glass or plastic cylinder) and a liquid phase which is the buffer (pH-stabilized salt water) running through. And you separate components based on which phase they’d rather hang out with.

A few common types used for proteins are affinity chromatography (AC), ion exchange chromatography (IEX) & size exclusion chromatography (SEC) and they use different resins. If the protein likes the resin (solid phase) more than it likes the liquid it came in with it’ll stick to the column. It might like the column because it’s oppositely-charged (this is the basis behind ION EXCHANGE CHROMATOGRAPHY (IEX)). The protein might also like the column more because it has some, more specific, special feature (like an engineered tag) that matches a special feature sticking off of the resin beads. This is how AFFINITY CHROMATOGRAPHY works. (note: the beads are usually porous – they have little tunnels running through them – and the affinity groups can stick out into these tunnels as well so you have more binding opportunities)

Both IEX & AC rely on the protein you want sticking to the column, while the other proteins flow through, then competing your protein off with salts and/or mimics or changing the pH to change the charge. But In SIZE EXCLUSION CHROMATOGRAPHY (SEC) you don’t want the protein to stick to the resin. Instead, you separate proteins by making smaller ones travel further because they can enter secret tunnels in the resin beads that big proteins can’t get into.

The theory behind these methods is the same whether you’re doing it with self-packed columns and gravity flow (which we still use all the time – more on this at the bottom) or the higher tech way with this fancy-dancey FPLC machine. Ours is an AKTA, and AKTA’s kinda like the “Google” of the protein chromatography world in that it basically dominates the market and if you say AKTA other protein-purifiers know what you mean (not to make anyone feel bad if they don’t! I didn’t know until I joined the hard-core chromatography crew – and I still get confused all the time by lingo from other fields!) 

In Fast Protein Liquid Chromatography (FPLC), a machine like the AKTA takes our protein sample and pumps it onto a column (which it’s gotten ready by flowing a bunch of buffer (pH-stable salt water) to “equilibrate” it. It then washes the column with the buffers we tell it to. And at the speeds we tell it to (FLOW RATE).

As the name implies, it *can* go fast, but you don’t always want it to or you’ll crush the resin in the column! Each column has different maximum flow rates. For the SEC columns I use, I typically run at ~0.7mL/min, which is actually pretty slow… And the fastest columns I run are only ~4mL/min. The times when the pumps are working their hardest is when doing pump washes. During those it’s pumping at 20mL/min, but it’s not going through any columns so you don’t have to worry about hurting them.

And, just in case, the system has pressure monitors at the entrance and exit of the columns. If the pre-column pressure (pressure going in) is too high it can damage the column hardware (the cylinder itself) & if the delta column pressure (difference between pressure going in & going out) is too high it could the resin in the column and/or the filter on top of the column are clogging up and generating dangerous pressure that can hurt the resin. So the AKTA will stop and alert you.

The liquid flows through lots of little tubes that offer different FLOW PATHS. Which path the liquid takes is dictated by lots and lots of valves to go with those lots and lots of little tubes. It’s kinda like a subway system that can change the tracks. So we can direct liquid into different columns and, when it comes out of the columns into the waste or a fractionater which collects it to “keep.”

It can collect different fraction sizes

  • for things like the flow-through in affinity chromatography, where your protein shouldn’t be there, you mainly collect the flow-through as a “just in case” so you don’t want to collect tons of tiny fractions, so we usually collect things like that in 50mL tubes
    • if you have a LOT of flow-through (like the other day when I was trying to get my protein to bind an anion exchange column but there was too much salt so I had to super dilute it and ended up having to flow almost 2L through a 5mL column) you can direct it through an “out” valve hooked up to a tube you can stick anywhere – like a giant graduated cylinder
  • when we’re doing the actual protein elution, we want to collect smaller portions for a couple reasons
    • 1) because the more concentrated it is the easier our life will be (and, as long as it’s not so concentrated that it clumps up (aggregates), proteins are usually happier when they’re more concentrated, so the next step is usually concentrating the eluted protein and the more concentrated it is to begin with, the less concentrating we’ll have to do
      • -for things like stepwise elutions, where you unstick everything in one fell swoop (e.g. go from no competitor to maximum competitor) 15mL tubes are good for this
    • 2) if proteins come off the column at different times but you collect everything that comes out in one tube, you’ve unpurified what you purified! But if you collect smaller portions, and different things are in different portions, you can keep separate the things you separate!  
      • for gradient elutions (work your way up to that max competitor concentration) depending on how tightly your protein’s stuck on, it’ll elute (get pushed off) at different concentrations – we often collect the elations in 96-well blocks. Each well can hold up to 2mL, but you can program the AKTA to do smaller fractions than that so you can be really picky about what you want to keep 

We choose which fractions we actually want to keep based on the CHROMATOGRAM. This is where we see the evidence of our protein coming out in the form of a peak in the 280nM wavelength absorbance. Proteins (in particular tryptophan, tyrosine, and phenylalanine) absorb that type of light so you can tell when protein’s elute because they “steal” that wavelength from the light spectrum. A UV MONITOR on the path between the bottom of the column & the fractionaer measures this. And the computer shows this to us as a peak. more here: https://bit.ly/2yzyi4w

We’re able to choose the flow rate because we control it with pumps. Yes, plural. We have 2 SYSTEM PUMPS so you can use 2 different buffers that send liquid first into a mixing chamber so you can mix them if you want to make a gradient for a gradient elution to introduce the “competitor” that will push your protein off the column (e.g. have a no salt & a high salt or a no imidazole & high imidazole (for His tags) you can mix). Or you can just use 1 for an “isocratic elution” like for SEC when you don’t need to change the buffer.

We also have another pump – a sample pump. This pump sucks up liquid through a sample line either until it reaches a volume we specify or until it senses air (hopefully because it’s sucked up all your sample and not because the line popped out…)

Another way to get a sample onto the column is through an INJECTION LOOP – kinda like people in line to get into a museum before it opens. – When you have a small volume to load, you can inject it into a loop of tubing that holds it until you want it to be let onto the column – then the injection valve opens up and lets your protein in.

FPLC can be used for tiny volumes (like when we inject samples into a 250uL loop) or huge volumes – industrial companies have huge columns that can handle liters and liters.

Another thing it has is a CONDUCTIVITY MONITOR to follow column equilibration (you want the conductivity to be stable before you put your sample on – this indicates that all the liquid in the column is the one you want your protein to meet) & salt gradient formation (as you add salt, the conductivity should increase – if it doesn’t, you have a problem somewhere… We have a pH MONITOR too.

Our lab has 4 AKTAs! (pinch me!) and we have an online “booking system” where we can sign up for when we want to use which one. Normally it’s not a problem to book it (and we don’t get charged or anything, the system’s just to “call it first”)

FPLC looks a lot like a related technique, HPLC. HPLC stands for High Performance Liquid Chromatography. HPLC uses higher pressures but lower flow rates. It’s usually used for small chemical compounds and sturdier beads that can withstand those high pressures.

The kind of chromatography we use is typically “preparatory” – we separate things to use them in their purer form. There’s also analytical chromatography, where you separate things just to see what’s there. this is similar to one of the differences between SEC & SDS-PAGE http://bit.ly/2xWH7VZ

There are many advantages to using the FPLC over relying on gravity to drip liquid through (gravity flow). My favorite reason – you don’t have to work in the cold room forever because it’s automated and, if you’re lucky, housed in a deli fridge not the cold room. When you’re working with precious proteins, you want to keep them safe. At higher temperatures, proteins can start to degrade especially if there are contaminating proteases (protein chewers) which at cold temps don’t have enough energy to chew your protein but do at higher temps. 

The 4th AKTA was a recent acquisition – and so was the deli fridge it’s housed in – and the fridge is having some issues so the temperature alarm is constantly going off even when the temp’s ok – but last year one of the fridges really did go down – and the temp went up – to like 30C! – and we had to move all the columns out but happily they seemed to survive unscathed!

But I don’t use the AKTA for everything. One of the nice things about gravity flow is that you can pack your own columns. AKTA pre-packed columns come in multiple sizes, but with gravity flow you can choose exactly what you want.

I prefer to use gravity flow for initial purification steps, where I’m working with “crude lysate” – the liquid you get when you break open cells (lyse them). Even though you then spin them down really fast (ultracentrifugation) to pellet the membrane pieces and other insoluble stuff and then you push it through a syringe filter, it can still be pretty viscous (syrupy) and this can clog up the AKTA lines. The AKTA’s really smart – it senses the buildup of pressure and slows down the flow rate – so it starts flowing REALLY SLOWLY. So much for that fast part… So it can be much more time-effective to go with gravity flow in these cases.

Even when it’s not crude lysate, you still need to worry about stuff clogging the tiny tubes – so we filter all of the buffers we send through them – usually using a bottle-top vacuum filter – it has a kind of “bowl” you screw on to the top of the bottle and you pour the buffer into the bowl and it gets sucked through a membrane into the bottle. 

Some practical advice:

  • Before starting an AKTA run, check all the tubing for bubbles. Air in the pumps can really mess up your runs, so you can manually prime the pumps with a syringe to pull through any trapped air. If you have the AKTA set to collect fixed fraction sizes, visually check after the fact to make sure that all the wells really do have the same volume. Variability could indicate air in the system. 
  • Remember to regularly empty the waste beaker and check that it doesn’t need emptying before you start an AKTA run – also, always have paper towels handy! 
  • Make sure the lines of your AKTA don’t pop out or they’ll start sucking in air not buffer/sample. If air gets into the pumps the peaks will go haywire, the pressure will flatline and the fraction sizes will be off if the line just pops out from one pump. Tape works nicely, and so do those caps with the little slits in them (don’t know the technical name)
  • you can use remote desktop software as a sort of “baby monitor” to keep an eye on the run from another room (or even from your bed) as it goes – good time for cleaning that bench mess… At first I felt sorry for my bench – it only ever gets a few minutes of unmessiness at a time – as soon as one’s cleaned up, another seems to magically appear… but now I think of it more as me allowing it to fulfill it’s life purpose of enabling scientific exploration – like on Friends where Phoebe’s opposed to trees being cut down to use as Christmas decorations, so Joey tells Phoebe that Christmas trees were “fulfilling their life purpose, by making people happy” 

Speaking of happiness – protein purifying makes me happy (as long as it’s not problematic!) but it can also be exhausting – and last week was one of those problematic, exhausting prep weeks – so I took some time to relax this weekend – including playing with LEGOs! I got this #nanoAKTA and it’s so fun! While AKTAs are for separating, the LEGO-ing should have been just putting things together – except I made a few boo-boos I had do quite a bit of separating!

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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