Size Exclusion Chromatography (SEC) (aka Gel Filtration): preparative & analytical on the AKTA

Of all the types of protein chromatography you may be usin’, size exclusion tends to cause the most confusion! Because, unlike in gel electrophoresis (e.g. SDS-PAGE), it’s the bigger proteins that win the gel mesh drag race when it comes to the size exclusion case! And, no matter what type of SEC (aka gel filtration) you’re doing (i.e. preparative or analytical), choosing the right column is critical!
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blog form (with full text & figures): bit.ly/sizeexc...
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text a shortened version of text from June
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SEC is a column-based method used to separate proteins by size/shape - either for:⠀
1. purification (preparative SEC), often as a “polishing step” after you’ve done other separations (e.g. affinity and/or ion exchange) or ⠀
2. to see if proteins interact (analytical SEC)⠀
Unlike with affinity chromatography and ion exchange chromatography, where the proteins actually bind (reversibly) to the resin (either because of some unique feature like an affinity tag (e.g. 6XHis or Strep) in the case of “affinity chromatography,” or because of charge in the case of ion exchange, with size exclusion chromatography (SEC, aka gel filtration (GF)) the proteins and beads don’t actually directly interact, they just take different routes. ⠀
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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. This chromatograph shows light absorption measured as the protein passes through a UV detector after coming off (eluting from) the column en route to a fraction collector.
You can think about it as having lots of cars traveling to the same destination - the fraction collector. The route has 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. Thus 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. ⠀
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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. From the protein’s perspective, they’re pretty “boring” as is - like well-paved roads they won’t slow the proteins down. ⠀
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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. On the bright side, at least those roads are less crowded, right? ⠀
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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. This is the basic of analytical size exclusion chromatography.
That’s what I’m doing today, but 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 a common technique for separating proteins in a gel slab to look (after staining) at the size & number of proteins in the sample. It’s 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 were just both present in the sample. bit.ly/sdspager... ⠀
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But with SEC, you don’t unfold the proteins, so protein-protein interactions can hold. The interactions have to be strong enough to withstand the journey. In analogy-terms, 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.⠀
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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) & the bigger at a smaller volume (shorter timepoint). When you mix them, the peaks will shift.⠀
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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, representing what was already in the beads before you started and what went through without going through any tunnels, depends on the column size.

[@YashapalSingh-m3r]

Can you please suggest us how to clean HiLoad Superdex 200 pg preparative SEC column? we are facing trouble with very high pressure for last two weeks, thanks. I am afraid to use sodium hydroxide.

[@thebumblingbiochemist]

Hi. I would just follow the guidance in the manual to make sure you don't do any damage to the column. Best of luck!

[@YashapalSingh-m3r]

@@thebumblingbiochemist Thanks for your suggestion. I contacted Cytiva and followed their suggestion. I have done a rigorous cleaning, I am hoping Akta will work fine from now onwards. Thanks again.

[@thebumblingbiochemist]

Glad you got help and hope it works well!

[@mpc7440]

Excellent explanation! Thanks.

[@LoomingShadows]

I'm doing this for the first time tomorrow, blessed release.

[@thebumblingbiochemist]

good luck!

[@zacharyw1558]

Great video, thank you for sharing your experience

[@thebumblingbiochemist]

Thanks! Glad you liked!

[@Cooking4me_928]

Love you explanation

[@thebumblingbiochemist]

Thank you!

[@huchang9794]

I have a question. I am doing analytical SEC and if I got the purified protein complex that is a protein complex by reconstituting a protein within a lipid nanodisc (and the belt MSP), can I see difference between the complex and the nanodisc in the bands of SDS page? Many thanks

[@thebumblingbiochemist]

I would think the SDS would break up the nanodisc

[@huchang9794]

I think so. so I will try blue native page@@thebumblingbiochemist

[@thebumblingbiochemist]

Best of luck!

[@huchang9794]

Thank you! I will keep you updated@@thebumblingbiochemist